scholarly journals Inhibition of TGF-β Signaling Suppresses Th17 Differentiation and Promotes Treg Numbers but Does Not Reduce Experimental Arthritis.

2021 ◽  
Author(s):  
Joyce Aarts ◽  
Arjan van Caam ◽  
Renoud M. Marijnissen ◽  
Monique M. Helsen ◽  
Birgitte Walgreen ◽  
...  
Keyword(s):  
T Cell ◽  
Ex Vivo ◽  
Joint Inflammation ◽  
Experimental Arthritis ◽  
T Cell Subsets ◽  
Murine Splenocytes ◽  
Th17 Differentiation ◽  
Synovial Explants

Abstract ObjectivesTGF-β is an important growth factor to promote the differentiation of T helper 17 (Th17) as well as regulatory T cells (Treg). Due to its dual role, the potential of TGF-β as therapeutic target in T cell-mediated diseases like rheumatoid arthritis (RA) is unclear. In this study, we investigated the effect of TGF-β inhibition on murine Th17 differentiation in vitro, on human RA synovial explants ex vivo, and on the development of experimental arthritis in vivo. MethodsMurine splenocytes were differentiated into Th17 cells, and the effect of the TGF-βRI inhibitor SB-505124 on Th17 differentiation was studied. RA synovial biopsies were cultured for 24h in the presence or absence of SB-505124. Experimental arthritis models were induced in C57Bl6 mice, and were treated daily with SB-505124. FACS analysis was performed to measure different T cell subsets. Histological sections were analysed to determine joint inflammation and destruction.ResultsSB-505124 potently reduced murine Th17 differentiation by decreasing Il7a and Rorc gene expression and IL-17 protein production. SB-505124 significantly suppressed IL-6 production by RA synovial explants. In the Th17-driven arthritis model, SB-505124 reduced Th17 levels, while increased levels of Tregs were observed. Despite this skewed Th17/Treg balance, SB-505124 treatment did not result in suppression of joint inflammation and destruction in this model.ConclusionsBlocking TGF-β signalling suppresses Th17 differentiation and improves the Th17/Treg balance. However, SB-505124 treatment does not suppress experimental arthritis, and is therefore not an adequate way to target Th17-driven inflammation.

scholarly journals Inhibition of TGF-β Signaling Suppresses Th17 Differentiation and Promotes Treg Numbers but does not Reduces Experimental Arthritis

2021 ◽  
Author(s):  
Joyce Aarts ◽  
Arjan Caam ◽  
Renoud M. Marijnissen ◽  
Monique M. Helsen ◽  
Birgitte Walgreen ◽  
...  
Keyword(s):  
T Cell ◽  
Ex Vivo ◽  
Joint Inflammation ◽  
Experimental Arthritis ◽  
T Cell Subsets ◽  
Murine Splenocytes ◽  
Th17 Differentiation ◽  
Synovial Explants

Abstract ObjectivesTGF-β is an important growth factor to promote the differentiation of T helper 17 (Th17) and regulatory T cells (Treg). The potential of TGF-β as therapeutic target in T cell-mediated diseases like rheumatoid arthritis (RA) is unclear. We investigated the effect of TGF-β inhibition on murine Th17 differentiation in vitro, on human RA synovial explants ex vivo, and on the development of experimental arthritis in vivo. MethodsMurine splenocytes were differentiated into Th17 cells, and the effect of the TGF-βRI inhibitor SB-505124 was studied. Synovial biopsies were cultured in the presence or absence of SB-505124. Experimental arthritis was induced in C57Bl6 mice and treated daily with SB-505124. FACS analysis was performed to measure different T cell subsets. Histological sections were analysed to determine joint inflammation and destruction.ResultsSB-505124 potently reduced murine Th17 differentiation by decreasing Il7a and Rorc gene expression and IL-17 protein production. SB-505124 significantly suppressed IL-6 production by synovial explants. In vivo, SB-505124 reduced Th17 levels, while increased levels of Tregs were observed. Despite this skewed Th17/Treg balance, SB-505124 treatment did not result in suppression of joint inflammation and destruction.ConclusionsBlocking TGF-β signalling suppresses Th17 differentiation and improves the Th17/Treg balance. However, SB-505124 treatment does not suppress experimental arthritis.

scholarly journals AB0082 INHIBITION OF TGFβ SIGNALING USING SB-505124 BLOCKS TH17 DIFFERENTIATION AND RESTORES THE TH17/TREG BALANCE IN VIVO, BUT DOES NOT SUPPRESS EXPERIMENTAL ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1341.1-1341
Author(s):  
J. Aarts ◽  
A. Van Caam ◽  
M. Helsen ◽  
B. Walgreen ◽  
E. Vitters ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protein Production ◽  
Joint Inflammation ◽  
Experimental Arthritis ◽  
Tgfβ Signaling ◽  
Th17 Differentiation ◽  
Synovial Explants

Background:TGFβ is an important growth factor that promotes the differentiation of T helper 17 (Th17) as well as regulatory T-cells (Treg). Due to its dual role, the potential of TGFβ as therapeutic target is unclear.Objectives:In this study we aimed to investigate the effect of inhibition of TGFβ signaling with the ALK5 inhibitor SB-505124 on human Th17 differentiationin vitro, on cytokine production by human rheumatoid arthritis (RA) synovial explants, and study the effects of local SB-505124 treatmentin vivoduring innate immune and Th17-driven experimental arthritis models.Methods:Magnetic sorted naïve human T cells were differentiated into Th17 cells with CD3/CD28 activation beads, IL-2, TGFβ, IL-1β, IL-23, αIFNƳ and αIL-4 for 6 days. Human RA synovial biopsies were cultured for 24h w/o 5µM SB-505124, and supernatant was analyzed by Luminex. T cell-independent SCW arthritis and Th17-driven IL-1/mBSA arthritis were induced in C57Bl6, and mice were treated with SB-505124 by daily intra-articular injections from day 0-4.Results:SB-505124 potently reduced human Th17 differentiationin vitroby decreasing IL-17 and RORƳt gene expression and IL-17 protein production. SB-505124 significantly suppressed IL-6 and TNFα protein production by human RA synovial explants. In addition, SB-505124 did not affect acute joint inflammation during SCW-arthritis (T-cell independent model). Interestingly, SB-505124 reduced Th17 levels in draining lymph nodes (dLN) during IL-1/mBSA arthritis while increased levels of Tregs were observed. Surprisingly, despite this skewed Th17/Treg balance, this did not result in suppression of joint inflammation and destruction in this Th17-driven arthritis model, whereas anti-IL-17 antibody treatment showed significant therapeutic effects.Conclusion:We revealed suppressive effects of SB-505124 on human Th17 differentiation and the Th17/Treg balance in arthritic mice. However, SB-505124 did not suppress joint inflammation and destruction. This indicates that despite the importance of TGFβ in Th17 differentiation, targeting TGFβ signaling is not enough to suppress experimental arthritis.Disclosure of Interests:None declared

Quantitative Assessment of the Impact of Ex Vivo Manipulation on the In Vivo Functionality of Human T Cells in RAG2−/− γc−/− mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 450-450
Author(s):  
Rozemarijn S. van Rijn ◽  
Elles R. Simonetti ◽  
Gert Storm ◽  
Mark Bonyhadi ◽  
Anton Hagenbeek ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
T Cell Subsets ◽  
Critical Parameters ◽  
In Vivo Evaluation ◽  
Human T Cells ◽  
The Impact

Abstract T cells retrovirally modified to express therapeutic genes encoding cytokines, exogenous TCRs or suicide molecules represent a novel class of immune therapeutics of great potency. However, recent clinical trials using retrovirally-modified T cells have indicated that T cells exhibit a diminished reactivity upon ex vivo manipulation. In addition, virus-specific memory T cells seem to be lost during gene transfer. In a BNML rat model we have shown that the culture procedure is one of the critical parameters. To preserve T cell reactivity, reliable models are required which permit readout of human T cell activity. We recently developed a huPBMC-RAG2−/−γc−/− mouse model for xenogeneic graft-versus-host disease (xGVHD), in which iv injection of 15 x 106 human T cells into RAG2−/−γc−/− mice consistently leads to high level engraftment and lethal xGVHD within 3 weeks in 80% of mice (van Rijn et al, Blood 2003). We have now used this model to analyze in vivo functionality of human T cells following different ex vivo culture procedures. For this, we cultured human T cells for 7 days with either of the two currently available clinically applicable stimulation conditions: 1) via CD3 and 2) via CD3/CD28. In addition, we included CD3/CD28/4-1BB stimulation to explore the effect of extensive costimulation. Mice were injected with escalating doses T cells. HuCD45+ cells in peripheral blood were measured by FACS. Lethal xGVHD occurred at only 6 times (90.106) the dose of fresh cells for CD3-stimulated T cells and 3 times for CD3/28- or CD3/28/4-1BB-stimulated cells. About 20% of surviving mice developed chronic xGVHD, independent of culture method. While lethal xGVHD was always associated with very high levels of engraftment (up to 95%) engraftment levels in chronic mice ranged from 1–75%. To compare the impact of the different culture conditions on in vivo T cell function, we analyzed engraftment potential. The fraction of huCD45+ cells was plotted against the time and the areas under the curves were compared. Based on a total of 68 mice, statistical analysis showed a 2-fold improvement of engraftment potential for C28-costimulated human T cells compared to CD3-stimulated cells (P<0.0001). Additional ligation of 4-1BB did not increase engraftment potential. In addition, different T cell subsets (naïve, memory, effector) were monitored based on the combined expression of CD45RA, CD27 and CCR7. For all primary T cells and variably cultured T cells, a strikingly similar pattern was observed in vivo. After 3 weeks mainly effector and memory effector T cells (both CD4+ and CD8+) could be detected, suggesting a (xeno-)antigen-driven survival and expansion. This was a very consistent observation independent of donor, culture condition, engraftment level or severity of disease. In conclusion, in vitro costimulation preserves in vivo functionality of human T cells and should therefore be included in future clinical protocols for ex vivo manipulation of T cells. These data show the feasibility to use the huPBMC-RAG2−/−γc−/− model for in vivo evaluation of in vitro effects on human T cells. This model is the most sensitive to date for in vivo evaluation of human T cells and will be a promising new tool for the study of human T cells in, for instance, autoimmune disease, cancer and infectious diseases like AIDS.

scholarly journals Brief Ex Vivo Incubation with Fas Ligand Selectively Depletes Alloreactive T Cells and Antigen Presenting Cells from Stem Cell Grafts

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2033-2033
Author(s):  
Hilit Levy-Barazany ◽  
Liat Pinkas ◽  
Galina Rodionov ◽  
Nitzan Marelly ◽  
Michal Tzadok ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Fas Ligand ◽  
Ex Vivo ◽  
T Cell Subsets ◽  
Ifn Γ ◽  
Antigen Presenting

Abstract Graft versus host disease (GvHD) proceeds to be the Achilles' heel of hematopoietic stem cell transplantation, with clinicians continue facing a classic conflict: too much GvHD and the patient is at risk for transplant-related mortality and decreased quality of life; too little GvHD and the patient is at increased risk of relapse of their malignant disease. T cells and antigen presenting cells (APCs) are major components of the hematopoietic G-CSF mobilized peripheral blood cells (PBCs) graft. While GvHD is T cell mediated, the APCs are required for the initiation and maintenance of the GvHD. To reduce the risk for GvHD, grafts are sometimes depleted of their T cells, however, while preventing GvHD, the critically important attributes of graft versus leukemia (GvL) effect and engraftment are reduced significantly. Novel strategies that aim to abrogate or ameliorate GvHD, while preserving engraftment and GvL are of great need. A short incubation (2hr) of G-CSF mobilized PBCs with multimeric Fas ligand (i.e. ApoGraft) selectively induces apoptosis in T cell subsets and APCs (Panels A and B), but not in CD34+ progenitor cells (data not shown). FasL treatment preferentially induces apoptosis in mature T cell subsets which express high levels of Fas (CD95), such as T stem cell memory (TSCM), T central memory (TCM), and T effector memory (TEM) cells, as well as the pro-inflammatory T cell subtypes TH1 and TH17 cells, while no apoptotic signal is detected in the non-expressing CD95 naïve T cells (Panel A). The expression of T cells and APCs activation markers; CD25 and HLA-DR, respectively, is significantly reduced following apoptotic challenge in vitro (Panel C), as well as in transplanted mice (data not shown). Furthermore, upon an activation stimulus with anti CD3/CD28 beads in vitro, ApoGraft derived T cells secrete lower levels of IFN-γ, than G-CSF mobilized PBCs derived T cells (Panel D). To gain deeper understanding of the kinetics of GvHD development in vivo, NSG mice were transplanted with ApoGraft or G-CSF mobilized PBCs. Homing, expansion and differentiation of human leukocytes subtypes within the mice bone marrow, spleen and blood, were monitored 3, 7 and 14 days post transplantation. Decreased levels of T and B cells infiltration and expansion were detected in the spleen (Panels E and F), suggesting reduced formation of allo-reactive T cell clones. Reduced proliferation of these cells was associated with lower levels of IFN-γ secreted to the plasma (Panel H) and was in correlation with reduced GvHD and prolonged survival of the ApoGraft transplanted mice (Panel G). Importantly, we have previously demonstrated both in-vitro and in-vivo that ApoGraft has similar GvL and stem cell engraftment capabilities, compared to control G-CSF mobilized PBCs (data not shown). In conclusion, in contrast to conventional T- cell depletion methods, ApoGraft, an ex-vivo FasL-treated graft, affects both the T-cells and APCs, leading to reduced GvHD, while maintaining GvL and engraftment potential (Panel I). ApoGraft is currently being evaluated in a Phase I/II clinical trial (NCT02828878) in subjects with hematologic malignancies undergoing matched related allo-HSCT. Figure. Figure. Disclosures Levy-Barazany: Cellect Biotherapeutics Ltd: Employment. Pinkas:Cellect Biotherapeutics Ltd: Employment. Rodionov:Cellect Biotherapeutics Ltd: Employment. Marelly:Cellect Biotherapeutics Ltd: Employment. Tzadok:Cellect Biotherapeutics Ltd: Employment. Bakimer:Cellect Biotherapeutics Ltd: Employment. Yarkoni:Cellect Biotherapeutics Ltd: Employment. Peled:Cellect Biotherapeutics Ltd: Consultancy. Zuckerman:Cellect Biotherapeutics Ltd: Consultancy.

696 Brentuximab vedotin, a CD30-directed antibody-drug conjugate, selectively depletes activated Tregs in vitro and in vivo

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A738-A738
Author(s):  
Bryan Grogan ◽  
Reice James ◽  
Michelle Ulrich ◽  
Shyra Gardai ◽  
Ryan Heiser ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Efflux Pump ◽  
Apoptotic Cell ◽  
T Cell Subsets ◽  
Memory Cells ◽  
Antibody Drug Conjugate ◽  
Selective Depletion

BackgroundRegulatory T cells (Tregs) play an important role in maintaining immune homeostasis, preventing excessive inflammation in normal tissues. In cancer, Tregs hamper anti-tumor immunosurveillance and facilitate immune evasion. Selective targeting of intratumoral Tregs is a potentially promising treatment approach. Orthogonal evaluation of tumor-infiltrating lymphocytes (TILs) in solid tumors in mice and humans have identified CCR8, and several tumor necrosis family receptors (TNFRs), including TNFSFR8 (CD30), as receptors differentially upregulated on intratumoral Tregs compared to normal tissue Tregs and other intratumoral T cells, making these intriguing therapeutic targets.Brentuximab vedotin (BV) is approved for classical Hodgkin lymphoma (cHL) across multiple lines of therapy including frontline use in stage III/IV cHL in combination with doxorubicin, vinblastine, and dacarbazine. BV is also approved for certain CD30-expressing T-cell lymphomas. BV is comprised of a CD30-directed monoclonal antibody conjugated to the highly potent microtubule-disrupting agent monomethyl auristatin E (MMAE).The activity of BV in lymphomas is thought to primarily result from tumor directed intracellular MMAE release, leading to mitotic arrest and apoptotic cell death.The role CD30 plays in normal immune function is unclear, with both costimulatory and proapoptotic roles described. CD30 is transiently upregulated following activation of memory T cells and expression has been linked to highly activated/suppressive IRF4+ effector Tregs.MethodsHere we evaluated the activity of BV on CD30-expressing T cell subsets in vitro and in vivo.ResultsTreatment of enriched T cell subsets with clinically relevant concentrations of BV drove selective depletion of CD30-expressing Tregs > CD30-expressingCD4+ T memory cells, with minimal effects on CD30-expressing CD8+ T memory cells. In a humanized xeno-GVHD model, treatment with BV selectively depleted Tregs resulting in accelerated wasting and robust T cell expansion. The observed differential activity on Tregs is likely attributable to significant increases in CD30 expression and reduced efflux pump activity relative to other T cell subsets. Interestingly, blockade of CD25 signaling prevents CD30 expression on T cell subsets without impacting proliferation, suggesting a link between CD25, the high affinity IL-2 receptor, and CD30 expression.ConclusionsTogether, these data suggest that BV may have an immunomodulatory effect through selective depletion of highly suppressive CD30-expressing Tregs.AcknowledgementsThe authors would like to thank Michael Harrison, PharmD for their assistance in abstract preparation.Ethics ApprovalAnimals studies were approved by and conducted in accordance with Seattle Genetics Institutional Care and Use Committee protocol #SGE-024.

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

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii111-ii111
Author(s):  
Lan Hoang-Minh ◽  
Angelie Rivera-Rodriguez ◽  
Fernanda Pohl-Guimarães ◽  
Seth Currlin ◽  
Christina Von Roemeling ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Ex Vivo ◽  
Adoptive T Cell Therapy ◽  
Whole Body ◽  
T Cell Therapy ◽  
Clinical Responses

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

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

2020 ◽  
Author(s):  
JL Reading ◽  
VD Roobrouck ◽  
CM Hull ◽  
PD Becker ◽  
J Beyens ◽  
...  
Keyword(s):  
T Cell ◽  
Treg Cell ◽  
Regulatory T Cell ◽  
Cellular Therapy ◽  
Ex Vivo ◽  
Fold Increase ◽  
Adoptive Cellular Therapy ◽  
T Cell Therapy

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

scholarly journals A novel BCMA/CD3 bispecific T-cell engager for the treatment of multiple myeloma induces selective lysis in vitro and in vivo

Leukemia ◽  
2016 ◽  
Vol 31 (8) ◽  
pp. 1743-1751 ◽  
Author(s):  
S Hipp ◽  
Y-T Tai ◽  
D Blanset ◽  
P Deegen ◽  
J Wahl ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cell ◽  
Plasma Cells ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Selective Lysis ◽  
Bispecific T Cell Engager

Abstract B-cell maturation antigen (BCMA) is a highly plasma cell-selective protein that is expressed on malignant plasma cells of multiple myeloma (MM) patients and therefore is an ideal target for T-cell redirecting therapies. We developed a bispecific T-cell engager (BiTE) targeting BCMA and CD3ɛ (BI 836909) and studied its therapeutic impacts on MM. BI 836909 induced selective lysis of BCMA-positive MM cells, activation of T cells, release of cytokines and T-cell proliferation; whereas BCMA-negative cells were not affected. Activity of BI 836909 was not influenced by the presence of bone marrow stromal cells, soluble BCMA or a proliferation-inducing ligand (APRIL). In ex vivo assays, BI 836909 induced potent autologous MM cell lysis in both, newly diagnosed and relapsed/refractory patient samples. In mouse xenograft studies, BI 836909 induced tumor cell depletion in a subcutaneous NCI-H929 xenograft model and prolonged survival in an orthotopic L-363 xenograft model. In a cynomolgus monkey study, administration of BI 836909 led to depletion of BCMA-positive plasma cells in the bone marrow. Taken together, these results show that BI 836909 is a highly potent and efficacious approach to selectively deplete BCMA-positive MM cells and represents a novel immunotherapeutic for the treatment of MM.

scholarly journals NIRF-Molecular Imaging with Synovial Macrophages-Targeting Vsig4 Nanobody for Disease Monitoring in a Mouse Model of Arthritis

2019 ◽  
Vol 20 (13) ◽  
pp. 3347 ◽  
Author(s):  
Fang Zheng ◽  
Siyu Luo ◽  
Zhenlin Ouyang ◽  
Jinhong Zhou ◽  
Huanye Mo ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Near Infrared ◽  
Single Photon ◽  
Ex Vivo ◽  
Photon Emission ◽  
Joint Inflammation ◽  
Experimental Arthritis ◽  
Emission Computed Tomography ◽  
Nirf Imaging

Nanobody against V-set and Ig domain-containing 4 (Vsig4) on tissue macrophages, such as synovial macrophages, could visualize joint inflammation in multiple experimental arthritis models via single-photon emission computed tomography imaging. Here, we further addressed the specificity and assessed the potential for arthritis monitoring using near-infrared fluorescence (NIRF) Cy7-labeled Vsig4 nanobody (Cy7-Nb119). In vivo NIRF-imaging of collagen-induced arthritis (CIA) was performed using Cy7-Nb119. Signals obtained with Cy7-Nb119 or isotope control Cy7-NbBCII10 were compared in joints of naive mice versus CIA mice. In addition, pathological microscopy and fluorescence microscopy were used to validate the arthritis development in CIA. Cy7-Nb119 accumulated in inflamed joints of CIA mice, but not the naive mice. Development of symptoms in CIA was reflected in increased joint accumulation of Cy7-Nb119, which correlated with the conventional measurements of disease. Vsig4 is co-expressed with F4/80, indicating targeting of the increasing number of synovial macrophages associated with the severity of inflammation by the Vsig4 nanobody. NIRF imaging with Cy7-Nb119 allows specific assessment of inflammation in experimental arthritis and provides complementary information to clinical scoring for quantitative, non-invasive and economical monitoring of the pathological process. Nanobody labelled with fluorescence can also be used for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Clonal Analyses of Retroviral Vector Integrations in ADA-SCID Patients Treated with Stem Cell Gene Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3249-3249
Author(s):  
Barbara Cassani ◽  
Grazia Andolfi ◽  
Massimiliano Mirolo ◽  
Luca Biasco ◽  
Alessandra Recchia ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Human Genome ◽  
Ex Vivo ◽  
Cd34 Cells

Abstract Gene transfer into hematopoietic stem/progenitor cells (HSC) by gammaretroviral vectors is an effective treatment for patients affected by severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA)-deficiency. Recent studied have indicated that gammaretroviral vectors integrate in a non-random fashion in their host genome, but there is still limited information on the distribution of retroviral insertion sites (RIS) in human long-term reconstituting HSC following therapeutic gene transfer. We performed a genome-wide analysis of RIS in transduced bone marrow-derived CD34+ cells before transplantation (in vitro) and in hematopoietic cell subsets (ex vivo) from five ADA-SCID patients treated with gene therapy combined to low-dose busulfan. Vector-genome junctions were cloned by inverse or linker-mediated PCR, sequenced, mapped onto the human genome, and compared to a library of randomly cloned human genome fragments or to the expected distribution for the NCBI annotation. Both in vitro (n=212) and ex vivo (n=496) RIS showed a non-random distribution, with strong preference for a 5-kb window around transcription start sites (23.6% and 28.8%, respectively) and for gene-dense regions. Integrations occurring inside the transcribed portion of a RefSeq genes were more represented in vitro than ex vivo (50.9 vs 41.3%), while RIS <30kb upstream from the start site were more frequent in the ex vivo sample (25.6% vs 19.4%). Among recurrently hit loci (n=50), LMO2 was the most represented, with one integration cloned from pre-infusion CD34+ cells and five from post-gene therapy samples (2 in granulocytes, 3 in T cells). Clone-specific Q-PCR showed no in vivo expansion of LMO2-carrying clones while LMO2 gene overexpression at the bulk level was excluded by RT-PCR. Gene expression profiling revealed a preference for integration into genes transcriptionally active in CD34+ cells at the time of transduction as well as genes expressed in T cells. Functional clustering analysis of genes hit by retroviral vectors in pre- and post-transplant cells showed no in vivo skewing towards genes controlling self-renewal or survival of HSC (i.e. cell cycle, transcription, signal transduction). Clonal analysis of long-term repopulating cells (>=6 months) revealed a high number of distinct RIS (range 42–121) in the T-cell compartment, in agreement with the complexity of the T-cell repertoire, while fewer RIS were retrieved from granulocytes. The presence of shared integrants among multiple lineages confirmed that the gene transfer protocol was adequate to allow stable engraftment of multipotent HSC. Taken together, our data show that transplantation of ADA-transduced HSC does not result in skewing or expansion of malignant clones in vivo, despite the occurrence of insertions near potentially oncogenic genomic sites. These results, combined to the relatively long-term follow-up of patients, indicate that retroviral-mediated gene transfer for ADA-SCID has a favorable safety profile.

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