Functional Verification of Novel ELMO1 Variants by Live Imaging in Zebrafish

ELMO1 (Engulfment and Cell Motility1) is a gene involved in regulating cell motility through the ELMO1-DOCK2-RAC complex. Contrary to DOCK2 (Dedicator of Cytokinesis 2) deficiency, which has been reported to be associated with immunodeficiency diseases, variants of ELMO1 have been associated with autoimmune diseases, such as diabetes and rheumatoid arthritis (RA). To explore the function of ELMO1 in immune cells and to verify the functions of novel ELMO1 variants in vivo, we established a zebrafish elmo1 mutant model. Live imaging revealed that, similar to mammals, the motility of neutrophils and T-cells was largely attenuated in zebrafish mutants. Consequently, the response of neutrophils to injury or bacterial infection was significantly reduced in the mutants. Furthermore, the reduced mobility of neutrophils could be rescued by the expression of constitutively activated Rac proteins, suggesting that zebrafish elmo1 mutant functions via a conserved mechanism. With this mutant, three novel human ELMO1 variants were transiently and specifically expressed in zebrafish neutrophils. Two variants, p.E90K (c.268G>A) and p.D194G (c.581A>G), could efficiently recover the motility defect of neutrophils in the elmo1 mutant; however, the p.R354X (c.1060C>T) variant failed to rescue the mutant. Based on those results, we identified that zebrafish elmo1 plays conserved roles in cell motility, similar to higher vertebrates. Using the transient-expression assay, zebrafish elmo1 mutants could serve as an effective model for human variant verification in vivo.

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Functional Verification of Novel ELMO1 Variants By Live Imaging in Zebrafish

Blood ◽

10.1182/blood-2021-148356 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3121-3121

Author(s):

Rongtao Xue ◽

Ying Wang ◽

Tienan Wang ◽

Mei Lyu ◽

Guiling Mo ◽

...

Keyword(s):

Cell Motility ◽

Conflicts Of Interest ◽

Live Imaging ◽

Dominant Negative ◽

Functional Verification ◽

Transient Expression Assay ◽

Dominant Negative Form ◽

Reduced Mobility ◽

Negative Form

Abstract ELMO1 (Engulfment and Cell Motility1) is a gene involved in regulating cell motility through the ELMO1-DOCK2-RAC complex. Contrary to DOCK2 (Dedicator of Cytokinesis 2) deficiency, which has been reported to be associated with immunodeficiency diseases, variants of ELMO1 have been associated with autoimmune diseases, such as diabetes and rheumatoid arthritis (RA). To explore the function of ELMO1 in immune cells and to verify the functions of novel ELMO1 variants in vivo, we established a zebrafish elmo1 mutant model. Live imaging revealed that similar to mammals, the motility of neutrophils and T-cells was largely attenuated in zebrafish mutants. Consequently, the response of neutrophils to injury or bacterial infection was significantly reduced in the mutants. Furthermore, the reduced mobility of neutrophils could be rescued by the expression of constitutively activated Rac proteins, suggesting that zebrafish elmo1 mutant functions via a conserved mechanism. With this mutant, three novel human ELMO1 variants were transiently and specifically expressed in zebrafish neutrophils. Two variants, p.E90K (c.268G>A) and p.D194G (c.581A>G) could efficiently recover the motility defect of neutrophils in the elmo1 mutant; however, the p.R354X (c.1060C>T) variant failed to rescue the mutant. Acts as a dominant-negative form, p.R354X (c.1060C>T) which failed to rescue the elmo1 mutant and inhibited neutrophil movement in siblings. Based on those results, we identified that zebrafish elmo1 played conserved roles in cell motility, similar to higher vertebrates. Using the transient-expression assay, zebrafish elmo1 mutants could serve as an effective model for human variant verification in vivo. Disclosures No relevant conflicts of interest to declare.

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Comparison of In Vitro and In Vivo Effects of Infliximab on Cytokine Production in Proliferating CD4+ T Cells in Patients with Rheumatoid Arthritis

Clinical Anti-Inflammatory & Anti-Allergy Drugs ◽

10.2174/2212703802666150127001412 ◽

2015 ◽

Vol 1 (2) ◽

pp. 122-128

Author(s):

Syuichi Koarada ◽

Yuri Sadanaga ◽

Natsumi Nagao ◽

Satoko Tashiro ◽

Rie Suematsu ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

T Cells ◽

Cd4 T Cells ◽

Cytokine Production

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102 Cord-blood derived NK cells, and CAR-T cells, an attractive improved immunotherapy treatment to be considered for hematological malignancies

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0102 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A113-A113

Author(s):

Mireia Bachiller García ◽

Lorena Pérez-Amill ◽

Anthony Battram ◽

Alvaro Urbano-Ispizua ◽

Beatriz Martín-Antonio

Keyword(s):

T Cells ◽

Tumor Cell ◽

Cord Blood ◽

Immune Cells ◽

In Vivo Models ◽

Cell Clusters ◽

Cytotoxicity Assays ◽

Anti Tumor Activity

BackgroundMultiple myeloma (MM) remains an incurable hematological malignancy where a proportion of patients relapse or become refractory to current treatments. Administration of autologous T cells modified with a chimeric antigen receptor (CAR) against B cell maturation antigen (BCMA) has achieved high percentages of complete responses. Unfortunately, the lack of persistence of CART-BCMA cells in the patient leads to relapses. On the other side, cord-blood derived natural killer cells (CB-NK) is an off-the-shelf cellular immunotherapy option to treat cancer patients with high potential due to their anti-tumor activity. However, clinical results in patients up to date have been sub-optimal. Whereas CB-NK are innate immune cells and their anti-tumor activity is developed in a few hours, CART cells are adaptive immune cells and their activity develops at later time points. Moreover, we previously described that CB-NK secrete inflammatory proteins that promote the early formation of tumor-immune cell clusters bringing cells into close contact and thus, facilitating the anti-tumor activity of T cells. Therefore, we hypothesized that the addition of a small number of CB-NK to CART cells would improve the anti-tumor activity and increase the persistence of CART cells.MethodsT cells transduced with a humanized CAR against BCMA and CB-NK were employed at 1:0.5 (CART:CB-NK) ratio. Cytotoxicity assays, activation markers and immune-tumor cell cluster formation were evaluated by flow cytometry and fluorescence microscopy. In vivo models were performed in NSG mice.ResultsThe addition of CB-NK to CART cells demonstrated higher anti-MM efficacy at low E:T ratios during the first 24h and in long-term cytotoxicity assays, where the addition of CB-NK to CART cells achieved complete removal of tumor cells. Analysis of activation marker CD69 and CD107a degranulation from 4h to 24h of co-culturing proved differences only at 4h, where CD69 and CD107a in CART cells were increased when CB-NK were present. Moreover, CB-NK accelerated an increased formation of CART-tumor cell clusters facilitating the removal of MM cells. Of note, CB-NK addition did not increase total TNFα and IFNγ production. Finally, an in vivo model of advanced MM with consecutive challenge to MM cells evidenced that the addition of CB-NK achieved the highest efficacy of the treatment.ConclusionsOur results suggest that the addition of ‘off-the-shelf’ CB-NK to CART cells leads to a faster and earlier immune response of CART cells with higher long-term maintenance of the anti-tumor response, suggesting this combinatorial therapy as an attractive immunotherapy option for MM patients.

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In vivo two-photon imaging of T cell motility in joint-draining lymph nodes in a mouse model of rheumatoid arthritis

Cellular Immunology ◽

10.1016/j.cellimm.2012.08.003 ◽

2012 ◽

Vol 278 (1-2) ◽

pp. 158-165 ◽

Cited By ~ 3

Author(s):

Tamás Kobezda ◽

Sheida Ghassemi-Nejad ◽

Tibor T. Glant ◽

Katalin Mikecz

Keyword(s):

Rheumatoid Arthritis ◽

T Cell ◽

Mouse Model ◽

Lymph Nodes ◽

Cell Motility ◽

Two Photon ◽

Photon Imaging ◽

Two Photon Imaging ◽

Draining Lymph Nodes

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708 Novel ways to exploit IL-21 to augment adoptive T cell transfer therapy against tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.708 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A737-A737

Author(s):

Anna Cole ◽

Guillermo Rangel RIvera ◽

Aubrey Smith ◽

Megan Wyatt ◽

Brandon Ware ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Immune Cells ◽

Striking Difference ◽

T Cell Therapy ◽

Cell Immunity ◽

Emory University

BackgroundIL-21 enhances the anti-tumor capacity of adoptively transferred CD8+ T cells, while IL-2 and IL-15 impair T cell immunity by driving their expansion to a more differentiated status. Yet, these cytokines can act on many different immune cells. Given the potency of IL-21, we tested if this cytokine directly augments T cells or rather if it enhances other immune cells in the culture that indirectly improves T cell therapy.MethodsTo test this question, splenocytes from pmel-1 transgenic mice were used, as all CD8+ T cells express a transgenic TCR specific for tumor-antigen gp10025–33 overexpressed on melanoma. We then peptide activated naïve CD8+ T cells enriched or not from the spleen of pmel-1 mice and expanded them in the presence of IL-21 or IL-2 (10 ng/mL) for four days. Expanded pmel-1 from these various cultures were then restimulated with irradiated splenocytes pulsed with gp10025–33 and grown an additional seven days with IL-2 (10 ng/mL), irrespective of their initial cytokine condition. The in vitro memory phenotype, exhaustion profile, and cytokine secretion of these cultures were then assayed. Furthermore, mice bearing B16KVP melanoma tumors were infused with pmel-1 T cells expanded via these various approaches and compared for their relative capacity to engraft, persist, and regress tumor in vivo.ResultsInterestingly, we discovered that IL-21-treated T cells generated from bulk splenocytes are phenotypically and functionally distinct from IL-21-treated isolated T cells. Upon restimulation, IL-21-treated T cells from bulk splenocytes exhibited an exhausted phenotype that was like anergic IL-2-treated T cells. Moreover, few cells expressed CD62L but expressed heightened markers of suppression, including TIM3, PD-1, and EOMES. Moreover, they produced more effector molecules, including granzyme B and IFN-gamma. In vivo IL-21-treated T cells expanded from bulk splenocytes engrafted and persisted poorly, in turn mediating suboptimal regression of melanoma. Conversely, IL-21 dramatically bolstered the engraftment and antitumor activity of T cells only if they were first isolated from the spleen prior to their expansion and infusion into the animal.ConclusionsCollectively, our data shows that IL-21 may improve ACT therapy best when used directly on antitumor CD8+ T cells. Further studies will illuminate the mechanism behind this striking difference and determine whether other cell subsets reactive to IL-21 cause T cell dysfunction and/or reduced bioavailability. These findings are important for defining the best culture conditions in which to use IL-21 for ACT.AcknowledgementsWe would like to acknowledge Emory University, The Winship Cancer Institute, and the Pediatrics/Winship Flow Cytometry Core.Ethics ApprovalAll animal procedures were approved by the Institutional Animal Care and Use Committee of Emory University, protocol number 201900225.

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Transcriptional heterogeneity in cancer-associated regulatory T cells is predictive of survival

10.1101/478628 ◽

2018 ◽

Cited By ~ 2

Author(s):

Nicholas Borcherding ◽

Kawther K. Ahmed ◽

Andrew P. Voigt ◽

Ajaykumar Vishwakarma ◽

Ryan Kolb ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Immune Cells ◽

Ex Vivo ◽

Expression Patterns ◽

Suppressive Effect ◽

Renal Clear Cell Carcinoma ◽

Cell Fates ◽

Treg Subset

Regulatory T cells (Tregs) are a population of T cells that exert a suppressive effect on a variety of immune cells and non-immune cells. The suppressive effects of Tregs are detrimental to anti-tumor immunity. Recent investigations into cancer-associated Tregs have identified common expression patterns for tumor-infiltration, however the functional heterogeneity in tumor-infiltrating (TI) Treg is largely unknown. We performed single-cell sequencing on immune cells derived from renal clear cell carcinoma (ccRCC) patients, isolating 160 peripheral-blood (PB) Tregs and 574 TI Tregs. We identified distinct transcriptional TI Treg cell fates, with a suppressive subset expressing CD177. We demonstrate CD177+ TI-Tregs have preferential suppressive effects in vivo and ex vivo. Gene signatures derived the CD177+ Treg subset had superior ability to predict survival in ccRCC and seven other cancer types. Further investigation into the development and regulation of TI-Treg heterogeneity will be vital to the application of tumor immunotherapies that possess minimal side effects.

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Tofacitinib Suppresses Several JAK-STAT Pathways in Rheumatoid Arthritis In Vivo and Baseline Signaling Profile Associates With Treatment Response

Frontiers in Immunology ◽

10.3389/fimmu.2021.738481 ◽

2021 ◽

Vol 12 ◽

Author(s):

Maaria Palmroth ◽

Krista Kuuliala ◽

Ritva Peltomaa ◽

Anniina Virtanen ◽

Antti Kuuliala ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

T Cells ◽

Treatment Response ◽

Peripheral Blood ◽

Janus Kinase ◽

Cytokine Signaling ◽

Stat3 Phosphorylation ◽

Stat Pathway

ObjectiveCurrent knowledge on the actions of tofacitinib on cytokine signaling pathways in rheumatoid arthritis (RA) is based on in vitro studies. Our study is the first to examine the effects of tofacitinib treatment on Janus kinase (JAK) - signal transducer and activator of transcription (STAT) pathways in vivo in patients with RA.MethodsSixteen patients with active RA, despite treatment with conventional synthetic disease-modifying antirheumatic drugs (csDMARDs), received tofacitinib 5 mg twice daily for three months. Levels of constitutive and cytokine-induced phosphorylated STATs in peripheral blood monocytes, T cells and B cells were measured by flow cytometry at baseline and three-month visits. mRNA expression of JAKs, STATs and suppressors of cytokine signaling (SOCS) were measured from peripheral blood mononuclear cells (PBMCs) by quantitative PCR. Association of baseline signaling profile with treatment response was also investigated.ResultsTofacitinib, in csDMARDs background, decreased median disease activity score (DAS28) from 4.4 to 2.6 (p < 0.001). Tofacitinib treatment significantly decreased cytokine-induced phosphorylation of all JAK-STAT pathways studied. However, the magnitude of the inhibitory effect depended on the cytokine and cell type studied, varying from 10% to 73% inhibition following 3-month treatment with tofacitinib. In general, strongest inhibition by tofacitinib was observed with STAT phosphorylations induced by cytokines signaling through the common-γ-chain cytokine receptor in T cells, while lowest inhibition was demonstrated for IL-10 -induced STAT3 phosphorylation in monocytes. Constitutive STAT1, STAT3, STAT4 and STAT5 phosphorylation in monocytes and/or T cells was also downregulated by tofacitinib. Tofacitinib treatment downregulated the expression of several JAK-STAT pathway components in PBMCs, SOCSs showing the strongest downregulation. Baseline STAT phosphorylation levels in T cells and monocytes and SOCS3 expression in PBMCs correlated with treatment response.ConclusionsTofacitinib suppresses multiple JAK-STAT pathways in cytokine and cell population specific manner in RA patients in vivo. Besides directly inhibiting JAK activation, tofacitinib downregulates the expression of JAK-STAT pathway components. This may modulate the effects of tofacitinib on JAK-STAT pathway activation in vivo and explain some of the differential findings between the current study and previous in vitro studies. Finally, baseline immunological markers associate with the treatment response to tofacitinib.

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Decreased MiR-128-3p alleviates the progression of rheumatoid arthritis by up-regulating the expression of TNFAIP3

Bioscience Reports ◽

10.1042/bsr20180540 ◽

2018 ◽

Vol 38 (4) ◽

Cited By ~ 11

Author(s):

Zhongbin Xia ◽

Fanru Meng ◽

Ying Liu ◽

Yuxuan Fang ◽

Xia Wu ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

T Cells ◽

Peripheral Blood ◽

Potential Role ◽

Enzyme Linked Immunosorbent Assay ◽

Normal Person ◽

Synovial Joint ◽

Peripheral Blood Mononuclear

Background: Rheumatoid arthritis (RA) is a inflammatory disease that characterized with the destruction of synovial joint, which could induce disability. Inflammatory response mediated the RA. It has been reported that MiR-128-3p is significantly increased in RA, while the potential role was still unclear. Methods: T cells in peripheral blood mononuclear cell (PBMC) were isolated from the peripheral blood from people of RA and normal person were used. Real-time PCR was performed to detect the expression of MiR-128-3p, while the protein expression of tumor necrosis factor-α-induced protein 3 (TNFAIP3) was determined using Western blot. The levels of IL-6 and IL-17 were measured using enzyme-linked immunosorbent assay (ELISA). The expression of CD69 and CD25 was detected using flow cytometry. The RA mouse model was constructed for verification of the role of MiR-128-3p. Results: The expression of MiR-128-3p was significantly increased, while TNFAIP3 was decreased, the levels of IL-6 and IL-17 were also increased in the T cells of RA patients. Down-regulated MiR-128-3p significantly suppressed the expression of p-IkBα and CD69, and CD25in T cells. MiR-128-3p targets TNFAIP3 to regulate its expression. MiR-128-3p knockdown significantly suppressed the activity of nuclear factor κB (NF-κB) and T cells by up-regulating TNFAIP3, while cells co-transfected with si-TNFAIP3 abolished the effects of MiR-128-3p knockdown. The in vivo experiments verified the potential role of MiR-128-3p on RA. Conclusion: Down-regulated MiR-128-3p significantly suppressed the inflammation response of RA through suppressing the activity of NF-κB pathway, which was mediated by TNFAIP3.

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Nicotinamide combined with gemcitabine is an immunomodulatory therapy that restrains pancreatic cancer in mice

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001250 ◽

2020 ◽

Vol 8 (2) ◽

pp. e001250

Author(s):

Benson Chellakkan Selvanesan ◽

Kiran Meena ◽

Amanda Beck ◽

Lydie Meheus ◽

Olaya Lara ◽

...

Keyword(s):

Pancreatic Cancer ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Immune Cells ◽

Mouse Tumor ◽

Epitope Spreading ◽

Stromal Compartment ◽

Positive Effects

BackgroundTreatments for pancreatic ductal adenocarcinoma are poorly effective, at least partly due to the tumor’s immune-suppressive stromal compartment. New evidence of positive effects on immune responses in the tumor microenvironment (TME), compelled us to test the combination of gemcitabine (GEM), a standard chemotherapeutic for pancreatic cancer, with nicotinamide (NAM), the amide form of niacin (vitamin B3), in mice with pancreatic cancer.MethodsVarious mouse tumor models of pancreatic cancer, that is, orthotopic Panc-02 and KPC (KrasG12D, p53R172H, Pdx1-Cre) grafts, were treated alternately with NAM and GEM for 2 weeks, and the effects on efficacy, survival, stromal architecture and tumor-infiltrating immune cells was examined by immunohistochemistry (IHC), flow cytometry, Enzyme-linked immunospot (ELISPOT), T cell depletions in vivo, Nanostring analysis and RNAscope.ResultsA significant reduction in tumor weight and number of metastases was found, as well as a significant improved survival of the NAM+GEM group compared with all control groups. IHC and flow cytometry showed a significant decrease in tumor-associated macrophages and myeloid-derived suppressor cells in the tumors of NAM+GEM-treated mice. This correlated with a significant increase in the number of CD4 and CD8 T cells of NAM+GEM-treated tumors, and CD4 and CD8 T cell responses to tumor-associated antigen survivin, most likely through epitope spreading. In vivo depletions of T cells demonstrated the involvement of CD4 T cells in the eradication of the tumor by NAM+GEM treatment. In addition, remodeling of the tumor stroma was observed with decreased collagen I and lower expression of hyaluronic acid binding protein, reorganization of the immune cells into lymph node like structures and CD31 positive vessels. Expression profiling for a panel of immuno-oncology genes revealed significant changes in genes involved in migration and activation of T cells, attraction of dendritic cells and epitope spreading.ConclusionThis study highlights the potential of NAM+GEM as immunotherapy for advanced pancreatic cancer.

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In Vivo Suppression of Autophagy via Lentiviral shRNA Targeting Atg5 Improves Lupus-Like Syndrome

BioMed Research International ◽

10.1155/2020/8959726 ◽

2020 ◽

Vol 2020 ◽

pp. 1-10

Author(s):

Chi-Jui Liu ◽

Shye-Jye Tang ◽

Chun-Che Chou ◽

Guang-Huan Sun ◽

Kuang-Hui Sun

Keyword(s):

T Cells ◽

Immune Cells ◽

Plasma Cells ◽

B Cell Activating Factor ◽

Dsdna Antibody ◽

Lymph Node Cells ◽

Complex Deposition ◽

Novel Strategy

In both mouse models and clinical patients with lupus, autophagy levels were significantly elevated and correlated with disease activity. Furthermore, autophagy can promote the survival of B and T cells, plasma cell differentiation, and antibody production. These results suggest that autophagy may promote the progression of lupus by regulating the survival of autoreactive immune cells. Therefore, we aimed at studying whether suppressing autophagy can modulate lupus progression in vivo. First, we found that the autophagy levels in splenocytes and lymphocytes of peripheral blood (PB) were elevated and positively correlated with disease severity in lupus-prone mice. The shAtg5-lentivirus, which effectively inhibits autophagy in vitro, was then injected into the lupus-prone mice. Autophagy levels in lymph node cells and PB lymphocytes were reduced following Atg5 suppression. We also found that lymphadenopathy and the numbers of plasma cells, CD4-CD8-, and CD4+ T cells decreased in mice treated with the shAtg5-lentivirus. The mice treated with shAtg5-lentivirus exhibited lower levels of proteinuria, serum anti-dsDNA antibody, B-cell activating factor (BAFF), and glomerular immune complex deposition. Therefore, targeting autophagy to moderate overactivated autophagy in immune cells seems to be a novel strategy for combination therapy of lupus.

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