Ex Vivo Expanded T Regulatory (Treg) Cells Block Conversion of Mixed Chimeras To Complete Donor Chimerism.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5168-5168
Author(s):  
Marina Lesnikova ◽  
Alla Nikitine ◽  
Nicola Mason ◽  
Richard A. Nash ◽  
George E. Georges
Keyword(s):  
T Cells ◽  
Ex Vivo ◽  
Treg Cells ◽  
Large Animal Model ◽  
Control Group ◽  
Large Animal ◽  
Not Given ◽  
Tolerance Mechanism ◽  
Donor Chimerism ◽  
Mixed Chimeras

Abstract A highly effective method to establish long-term, stable mixed hematopoietic chimerism was developed in the dog model. This involves nonmyeloablative allogeneic hematopoietic cell transplantation (HCT), consisting of 2 Gray (Gy) total body irradiation (TBI), dog leukocyte antigen (DLA)-identical marrow, and short-term post-grafting immunosuppression. We hypothesized that CD4+CD25+ Treg cells may be important regulators for the maintenance of cellular immune tolerance after allogeneic HCT. Previously, we showed in 8 mixed chimeras that naive donor lymphocyte infusion (DLI) did not change the level of donor chimerism. However, reconditioning mixed chimeras with 2 Gy TBI followed by DLI “breaks” tolerance and increases the level of donor chimerism. Seven mixed chimeras were reconditioned with 2 Gy TBI followed by DLI. Within 4 weeks after DLI, conversion to 100% donor chimerism was seen in 5 of 7 dogs and 2 dogs had a > 50% sustained increase in donor chimerism. Four recipients developed graft-versus host disease (GVHD). A control group of 3 mixed chimeras reconditioned with 2 Gy TBI without DLI had no change in donor chimerism. These results suggest that reconditioning with 2 Gy TBI followed by DLI can break the tolerance mechanism established in mixed chimeras. Next we asked if CD4+CD25+ Treg cells obtained from mixed chimeras before reconditioning could block the increase in donor chimerism following 2 Gy TBI and DLI. Peripheral blood mononuclear cells (PBMC) from 8 mixed chimeras were obtained by leukapheresis and cultured in bulk mixed leukocyte culture (MLC) with 3rd party DLA-mismatched, unrelated and irradiated CD34+ derived dendritic cells (10:1 responder: stimulator ratio) or PBMC (1:1). On day 4 of MLC, CD25+ cells were isolated by positive immunomagnetic selection. Next, artificial antigen presenting cells (aAPC, KT32) were added to expand the CD4+CD25+ Treg cells. The aAPC expressed Fcγ receptor CD32, canine CD86, and human IL-15, were loaded with the canine-specific mitogenic anti-CD3ε antibody 17.6F9 and irradiated prior to stimulation of CD4+CD25+ Treg. After 7 days, Treg were expanded a median of 23 (range, 8–36)-fold. Expanded CD4+CD25+ Treg were assessed for phenotype and in vitro function. The Treg cells were generated from 8 mixed chimeras and were infused back into the respective dogs (median dose 1× 107/kg) after reconditioning with 2 Gy TBI and immediately prior to DLI. In 6 of 8 dogs there was no change in the level of donor chimerism at 16–20 weeks follow-up; 2 dogs converted to complete donor chimerism within 6 weeks. Treatment with expanded Treg cells blocked conversion to complete donor chimerism after 2 Gy TBI +DLI in 6 of 8 dogs, compared with significant increases in donor chimerism for all 7 dogs after 2 Gy TBI +DLI not given Treg (p=.007). None of the 8 Treg recipient dogs developed GVHD, compared with 4 of 7 not given Treg, (p=0.02). A control group of 4 mixed chimeras were infused with expanded, non-Treg CD25− T cells. To this end, CD25+ T cells were immunomagneticaly depleted on day 4 of MLC. CD25− T cells were expanded with aAPC. Three of 4 dogs converted to the complete donor chimerism within 7 weeks after 2 Gy TBI, non-Treg and DLI. These results suggest that ex vivo expanded CD4+CD25+ Treg cells have in vivo function in a large animal model and can restore the tolerance mechanism in mixed chimeras that is broken by 2 Gy TBI and DLI.

scholarly journals The safety and efficacy of BCG encapsulated alginate particle (BEAP) against M.tb H37Rv infection in Macaca mulatta : A pilot study

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ashwani Kesarwani ◽  
Parul Sahu ◽  
Kshama Jain ◽  
Prakriti Sinha ◽  
K. Varsha Mohan ◽  
...  
Keyword(s):  
T Cells ◽  
Macaca Mulatta ◽  
Ex Vivo ◽  
Protective Efficacy ◽  
Rhesus Macaques ◽  
Allergic Response ◽  
Control Group ◽  
Activated T Cells ◽  
Primate Model ◽  
Safety And Efficacy

AbstractDue to the limited utility of Bacillus Calmette–Guérin (BCG), the only approved vaccine available for tuberculosis, there is a need to develop a more effective and safe vaccine. We evaluated the safety and efficacy of a dry powder aerosol (DPA) formulation of BCG encapsulated alginate particle (BEAP) and the conventional intradermal BCG immunization in infant rhesus macaques (Macaca mulatta). The infant macaques were immunized intratracheally with DPA of BEAP into the lungs. Animals were monitored for their growth, behaviour, any adverse and allergic response. The protective efficacy of BEAP was estimated by the ex-vivo H37Rv infection method. Post-immunization with BEAP, granulocytes count, weight gain, chest radiography, levels of liver secreted enzymes, cytokines associated with inflammation like TNF and IL-6 established that BEAP is non-toxic and it does not elicit an allergic response. The T cells isolated from BEAP immunized animals’ blood, upon stimulation with M.tb antigen, secreted high levels of IFN-γ, TNF, IL-6 and IL-2. The activated T cells from BEAP group, when co-cultured with M.tb infected macrophages, eliminated largest number of infected macrophages compared to the BCG and control group. This study suggests the safety and efficacy of BEAP in Non-human primate model.

scholarly journals FOXP3 expression accurately defines the population of intratumoral regulatory T cells that selectively accumulate in metastatic melanoma lesions

Blood ◽  
2008 ◽  
Vol 112 (13) ◽  
pp. 4953-4960 ◽  
Author(s):  
Mojgan Ahmadzadeh ◽  
Aloisio Felipe-Silva ◽  
Bianca Heemskerk ◽  
Daniel J. Powell ◽  
John R. Wunderlich ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Microenvironment ◽  
Metastatic Melanoma ◽  
Treg Cell ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Production Capacity ◽  
Biological Marker ◽  
Foxp3 Expression ◽  
Treg Cells

Abstract Regulatory T (Treg) cells are often found in human tumors; however, their functional characteristics have been difficult to evaluate due to low cell numbers and the inability to adequately distinguish between activated and Treg cell populations. Using a novel approach, we examined the intracellular cytokine production capacity of tumor-infiltrating T cells in the single-cell suspensions of enzymatically digested tumors to differentiate Treg cells from effector T cells. Similar to Treg cells in the peripheral blood of healthy individuals, tumor-infiltrating FOXP3+CD4 T cells, unlike FOXP3− T cells, were unable to produce IL-2 and IFN-γ upon ex vivo stimulation, indicating that FOXP3 expression is a valid biological marker for human Treg cells even in the tumor microenvironment. Accordingly, we enumerated FOXP3+CD4 Treg cells in intratumoral and peritumoral sections of metastatic melanoma tumors and found a significant increase in proportion of FOXP3+CD4 Treg cells in the intratumoral compared with peritumoral areas. Moreover, their frequencies were 3- to 5-fold higher in tumors than in peripheral blood from the same patients or healthy donors, respectively. These findings demonstrate that the tumor-infiltrating CD4 Treg cell population is accurately depicted by FOXP3 expression, they selectively accumulate in tumors, and their frequency in peripheral blood does not properly reflect tumor microenvironment.

scholarly journals Cellular and acellular ex vivo lung perfusion preserve functional lung ultrastructure in a large animal model: a stereological study

2018 ◽  
Vol 19 (1) ◽  
Author(s):  
Jasmin Steinmeyer ◽  
Simon Becker ◽  
Murat Avsar ◽  
Jawad Salman ◽  
Klaus Höffler ◽  
...  
Keyword(s):  
Animal Model ◽  
Ex Vivo ◽  
Lung Perfusion ◽  
Large Animal Model ◽  
Large Animal ◽  
Ex Vivo Lung Perfusion ◽  
Lung Ultrastructure

Abstract 657: A New Translational Model of Hypercholesterolemia and Atherosclerosis: Effect of Statins on LDLR KO Miniature Swine

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Dale E Mais ◽  
Thomas Vihtelic ◽  
Chidozie Amuzie ◽  
Steven Denham ◽  
John R Swart ◽  
...  
Keyword(s):  
High Fat Diet ◽  
Clinical Chemistry ◽  
Small Animal ◽  
Normal Diet ◽  
Large Animal Model ◽  
Control Group ◽  
Large Animal ◽  
Wild Type ◽  
High Fat ◽  
Miniature Swine

Small animal models of atherosclerosis are commonly used in drug studies; however, the results often fail to translate into the clinic. A large animal model that more accurately reflects the human disease is needed. We recently developed a transgenic Yucatan pig model in which the LDL receptor (LDLR) gene is knocked out. Five groups of Yucatan pigs (N=4 per group), either wild type (LDLR+/+) or heterozygote (LDLR+/-) were fed a normal diet or a high fat diet for a six month period. One of the heterozygote/high fat diet groups in addition received a daily dose of a statin (atorvastatin) at 3 mg/kg. Every two weeks during the study a variety of clinical chemistry parameters were measured. At study termination, select arteries were collected, stained for lipid deposits and quantitated. In addition, sections of these arteries were prepared for immunohistochemistry to detect selected markers of macrophage infiltration into the atherosclerotic plaques. As expected, pigs fed a high fat diet gained significantly more weight at six months whether they were wild type or LDLR+/-. Atorvastatin appeared to attenuate this weight gain. There were significant increases in total cholesterol, HDL and LDL in pigs fed the high fat diet compared to their corresponding control group. The group receiving the atorvastatin had reduced values of these parameters compared to controls showing that a statin had a beneficial effect on lipid levels even in a high fat diet scenario. VLDL levels were not affected but there were triglyceride changes across the groups. Liver function was unchanged based on total bilirubin and AST while ALT measurements were altered in some of the groups. Immunohistochemistry and histomorphometry was performed on some arteries. Atorvastatin-induced amelioration of hypercholesterolemia in this model underscores its translational utility.

scholarly journals Light-degradable hydrogels as dynamic triggers for gastrointestinal applications

2020 ◽  
Vol 6 (3) ◽  
pp. eaay0065 ◽  
Author(s):  
Ritu Raman ◽  
Tiffany Hua ◽  
Declan Gwynne ◽  
Joy Collins ◽  
Siddartha Tamang ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Implantable Devices ◽  
Large Animal Model ◽  
Large Animal ◽  
Gi Tract ◽  
Biomedical Device ◽  
Precise Tool ◽  
And Performance

Triggerable materials capable of being degraded by selective stimuli stand to transform our capacity to precisely control biomedical device activity and performance while reducing the need for invasive interventions. Here, we describe the development of a modular and tunable light-triggerable hydrogel system capable of interfacing with implantable devices. We apply these materials to two applications in the gastrointestinal (GI) tract: a bariatric balloon and an esophageal stent. We demonstrate biocompatibility and on-demand triggering of the material in vitro, ex vivo, and in vivo. Moreover, we characterize performance of the system in a porcine large animal model with an accompanying ingestible LED. Light-triggerable hydrogels have the potential to be applied broadly throughout the GI tract and other anatomic areas. By demonstrating the first use of light-degradable hydrogels in vivo, we provide biomedical engineers and clinicians with a previously unavailable, safe, dynamically deliverable, and precise tool to design dynamically actuated implantable devices.

Allograft T Cell Content Influences Early Engraftment after Reduced-Intensity Stem Cell Transplantation (RIST).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3662-3662
Author(s):  
Robert M. Dean ◽  
Daniel H. Fowler ◽  
Nancy M. Hardy ◽  
Jeanne Odom ◽  
Kathleen Castro ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Group Iii ◽  
Donor Chimerism ◽  
Group I ◽  
Gvhd Prophylaxis ◽  
Donor T Cells ◽  
Reduced Intensity

Abstract Allogeneic hematopoietic stem cells (HSC) generally engraft rapidly and completely after myeloablative conditioning. However, with reduced-intensity conditioning (RIC), mixed chimerism and graft failure are more common. Host immune status and HSC number are factors known to affect engraftment after reduced-intensity stem cell transplantation (RIST). In addition, donor T cells within the allograft may also influencethe kinetics of donor engraftment after RIST. To evaluate this, we performed a controlled comparison of engraftment outcomes among 3 groups undergoing RIST, varying by ex vivo T cell depletion (TCD) or in vivo depletion of activated T cells with methotrexate (MTX) to prevent graft-versus-host disease (GVHD). Group I (n = 50) received T cell replete (TCR) peripheral blood stem cells (PBSC) with cyclosporine (CSA) alone for GVHD prophylaxis. Group II (n = 17) received ex vivo TCD PBSC (positive/negative selection with T cell add-back to uniform dose of 1 x 105 CD3+ cells/kg) with CSA alone for GVHD prophylaxis. Group III (n = 31) received TCR PBSC with CSA plus MTX (5 mg/m2 IV x 4 doses) for GVHD prophylaxis. The 3 groups were similarly immunosuppressed from prior therapy before RIST (median absolute lymphocyte counts 330/μL, 260/μL, and 307/μL for Groups I, II, and III, respectively), and received an identical RIC regimen (fludarabine/cyclophosphamide) plus comparable numbers of filgrastim-mobilized PBSC from HLA-matched sibling donors (median 7.9 x 106, 7.6 x 106, and 6.8 x 106 CD34+ cells/kg, respectively; median 3.6 x 108, 1.0 x 105, and 3.2 x 108 CD3+ cells/kg, respectively). Hematopoietic recovery was slowest in Group III, consistent with the myelosuppressive effects of MTX (Table). A greater proportion of patients in Group I achieved complete donor chimerism (≥ 95%) by day +28 than in Groups II or III (P < 0.025), and at day +100, mixed donor chimerism persisted more often in Groups II and III than in Group I patients (P < 0.01). Correspondingly, early (< day +42) occurrence of grade 3–4 acute GVHD, before initiation of planned sequential donor lymphocyte infusions (DLI) in Group II, was more frequent in Group I than in either Groups II or III (p=0.08). Table: Hematopoietic Recovery, Engraftment, and GVHD Group Days to ANC > 500, median (range) Days to plt > 100, median (range) Donor chimerism ≥ 95% Early acute GVHD, grades 3–4 Day +28 Day +100 I 9 (7–13) 15.5 (12-42) 37/44 (84%) 36/38 (95%) 9/50 (18%) II 9 (7–10) 17.5 (11–40) 8/17 (47%) 9/14 (65%) 0/17 (0%) III 14 (7–21) 21.5 (12–85) 23/31 (74%) 21/31 (68%) 2/31 (6%) Thus, the deletion of T cells by either ex vivo TCD or in vivo MTX administration measurably alters the kinetics and degree of donor T cell engraftment after RIST. These observations provide evidence that donor T cells are an independent factor affecting engraftment of allogeneic HSC after RIST by compensating for incomplete host immune ablation. These data also support the hypothesis that a graft-versus-host effect plays a significant role in engraftment after RIST. Manipulation of donor T cells through graft engineering techniques may be a useful strategy to enhance engraftment in the setting of RIST.

TNFR25 Expression on CD4+CD25+ T Cells: Down Modulation of Regulatory Activity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3165-3165
Author(s):  
Vadim Deyev ◽  
Melinda Roskos ◽  
Robert B. Levy ◽  
Eckhard R. Podack
Keyword(s):  
T Cells ◽  
Cell Function ◽  
Ex Vivo ◽  
Cell Activity ◽  
Treg Cells ◽  
Regulatory Activity ◽  
T Regulatory Cell ◽  
Tnf Receptor Family ◽  
Receptor Family

Abstract TNFR25 (“DR3”) is a member of the TNF receptor family that is expressed by activated CD4+ and CD8+ T cells. To determine if activated CD4+CD25+ T cells also expressed this TNFR family molecule, B6 CD4+CD25+ T cells were stimulated with anti-CD3/CD28 coated beads (kind gift of Dr. B. Blazar, U. Minn.) and expanded for 3–4 days. TNFR25 expression was readily detected on CD4+CD25+ FoxP3+ T cells. Since other members of the TNF receptor family (GITR, OX40, 4–1BB) are known to influence T regulatory cell function, we investigated whether TNFR25 signaling can regulate CD4+CD25+ T cell activity. TNFR25 triggering in B6-wt T regulatory CD4+CD25+ cells with the recombinant TNFR25 ligand TL1A or agonistic anti-TNFR25 antibody (4C12) resulted in reduction of their ability to suppress anti-CD3 induced ex-vivo proliferation of CD4+CD25− cells. 4C12 mediated TNFR25 signaling also reduced B6-wt Treg mediated inhibition of peptide induced proliferation of OVA-specific B6 CD8+ (OT-I) cells. To further investigate a role for TNFR25 in Treg cell regulation, TNFR25 (full length) transgenic mice were generated and bred onto the BL/6 background. CD4+CD25+ cells from these TNFR25 tg mice were found to possess diminished T regulatory activity in vitro as determined by their diminished inability to regulate proliferation by B6-wt CD4+ and OT-I CD8+ T cells. To assess their in vivo regulatory activity, B6-wt and B6 TNFR25 tg Treg cells were examined for their ability to inhibit graft vs. host disease (GVHD) following allogeneic MHC class I/II mismatched BMT. In contrast to B6-wt Treg cells, TNFR25 tg Treg cells exhibited significantly diminished ability to regulate the onset of GVHD in vivo as assessed by weight loss and clinical symptoms. Using agonistic antibody, stimulation of TNFR25 on transgenic Treg cells was also found to effectively remove the ex-vivo regulatory activity expressed by this population. To exclude any possible direct co-stimulatory effects of 4C12 antibody on the responding proliferating cells, CD4+CD25−T cells from TNFR25 dominant negative transgenic mice were employed. 4C12 mab again abolished Treg cell inhibitory activity. The effect of TNFR25 agonists on T reg cell activity in vivo is being further investigated in both mouse models of GVHD and IBD diseases. Initial observations administering 4C12 post-allogeneic BMT together with B6-wt Treg cells indicate a reduction in their ability to regulate GVHD. In total, these studies identify TNFR25 as a new potential target for augmenting CD4+ and CD8+ responses by concomitant direct co-stimulation of effecter cells and inhibition of T regulatory cell function.

scholarly journals Vasoactive intestinal peptide generates human tolerogenic dendritic cells that induce CD4 and CD8 regulatory T cells

Blood ◽  
2006 ◽  
Vol 107 (9) ◽  
pp. 3632-3638 ◽  
Author(s):  
Elena Gonzalez-Rey ◽  
Alejo Chorny ◽  
Amelia Fernandez-Martin ◽  
Doina Ganea ◽  
Mario Delgado
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Vasoactive Intestinal Peptide ◽  
Ex Vivo ◽  
Inflammatory Diseases ◽  
Treg Cells ◽  
Peripheral Tolerance ◽  
Blood Monocytes ◽  
Endogenous Factors ◽  
Tolerogenic Dcs

Induction of antigen-specific tolerance is critical for autoimmunity prevention and immune tolerance maintenance. In addition to their classical role as sentinels of the immune response, dendritic cells (DCs) play important roles in maintaining peripheral tolerance through the induction/activation of regulatory T (Treg) cells. The possibility of generating tolerogenic DCs opens new therapeutic perspectives in autoimmune/inflammatory diseases. Characterizing endogenous factors that contribute to the development of tolerogenic DCs is highly relevant. We here report that the immunosuppressive neuropeptide vasoactive intestinal peptide (VIP) induces the generation of human tolerogenic DCs with the capacity to generate CD4 and CD8 Treg cells from their respective naive subsets. The presence of VIP during the early stages of DC differentiation from blood monocytes generates a population of IL-10-producing DCs unable to fully mature after the effects of inflammatory stimuli. CD4 Treg cells generated with VIP-differentiated DCs resemble the previously described Tr1 cells in terms of phenotype and cytokine profile. CD8 Treg cells generated with tolerogenic VIP DCs have increased numbers of IL-10-producing CD8+CD28--CTLA4+ T cells. CD4 and CD8 Treg cells primarily suppress antigen-specific TH1-mediated responses. Therefore, the possibility of generating or expanding ex vivo tolerogenic DCVIPs opens new therapeutic perspectives for treating autoimmune diseases and graft-versus-host disease after allogeneic transplantation in humans.

scholarly journals Ex VivoFluorescence Molecular Tomography of the Spine

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Monish Pimpalkhare ◽  
Jin Chen ◽  
Vivek Venugopal ◽  
Xavier Intes
Keyword(s):  
Ex Vivo ◽  
Imaging Study ◽  
Intervertebral Disk ◽  
Large Animal Model ◽  
Data Sets ◽  
Large Animal ◽  
Wide Field ◽  
Accurate Localization ◽  
Quantification Accuracy ◽  
The Impact

We investigated the potential of fluorescence molecular tomography to imageex vivosamples collected from a large animal model, in this case, a dog spine. Wide-field time-gated fluorescence tomography was employed to assess the impact of multiview acquisition, data type, and intrinsic optical properties on the localization and quantification accuracy in imaging a fluorescent inclusion in the intervertebral disk. As expected, the TG data sets, when combining early and late gates, provide significantly better performances than the CW data sets in terms of localization and quantification. Moreover, the use of multiview imaging protocols led to more accurate localization. Additionally, the incorporation of the heterogeneous nature of the tissue in the model to compute the Jacobians led to improved imaging performances. This preliminary imaging study provides a proof of concept of the feasibility of quantitatively imaging complexex vivosamples nondestructively and with short acquisition times. This work is the first step towards employing optical molecular imaging of the spine to detect and characterize disc degeneration based on targeted fluorescent probes.

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