Third Party Cytokine-Induced Killer Cells Eliminate Highly Activated T-Cells, Leading to Protect from Murine Lethal Graft-Versus-Host Disease

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2158-2158
Author(s):  
Rie Kuroda ◽  
Shintaro Mase ◽  
Hideaki Maeba ◽  
Toshihiro Fujiki ◽  
Masaki Fukuda ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Acute Gvhd ◽  
Third Party ◽  
Activated T Cells ◽  
Graft Versus Host ◽  
Cik Cells ◽  
Alloreactive T Cells ◽  
Antigen Presenting

Abstract Protecting from lethal graft-versus-host disease (GVHD) while preserving graft-versus-tumor effects by adoptive immunotherapy would be an ideal goal in allogeneic hematopoietic stem cell transplantation (HSCT), especially using third party derived cells, because the use of third party cells would open a huge source of availability and feasibility across major histocompatibility barriers. We have demonstrated that third party cytokine-induced killer (CIK) cells protected from murine lethal GVHD with faster hematological recovery, which are ex vivo-expanded T lymphocytes expressing both natural killer and T-cell markers with strong cytotoxic activity both against autologous and allogeneic tumor cells. However the mechanism responsible for ameliorating acute GVHD by third party CIK cells are still unknown. First, lethally irradiated Balb/c (H-2d) mice were given C3H (H-2k) bone marrow cells with C3H whole splenocytes to induce lethal GVHD with/without third party (C57/BL6 (H-2b)) CIK cells or third party whole splenocytes on day 0 or day4. Mice receiving third party CIK cells showed much less GVHD and significant survival benefit compared those with third party splenocytes as shown below (p<0.05). On day7 after BMT, the percentage of CD69 on CD8 T-cells in peripheral blood (PB) in the mice receiving third party CIK cells was significantly lower than those in GVHD control mice as shown below (p<0.05), meanwhile donor derived not-activated T-cells were preserved in both CD4 and CD8 T-cells. From these results, we hypothesized that third party CIK cells prevent acute GVHD by selectively depleting highly proliferative alloreactive T-cells stimulated early after transplant, while sparing naïve T-cells. To test this, we performed mixed lymphocyte reaction (MLR) by adding third party CIK cells or third party splenocytes (Stimulator: Balb/c splenocytes, Responder: C3H splenocytes, third party cells: C57/BL6). The percentage of CD69 on responder derived CD8 T-cells in the culture mixture with third party CIK cells was much less compared to that with third party splenocytes as shown below (p<0.05). We have previously shown that allogeneic CIK cells had cytotoxicity against cultured dendritic cells (DCs) by 51Cr release assay. Therefore reduced alloreaction by third party CIK cells might be due in part to the elimination of antigen presenting cells such as DCs. Next, to demonstrate that third party CIK cells could eliminate responder cells directly and then reduce the alloreaction, we performed MLR like experiments using PHA to activate T-cells strongly instead of antigen presenting cells as a stimulator. As shown below, we have demonstrated that the percentage of highly activated T-cells (CD8+CD69+) in third party CIK added plates was significantly lower compared to that in third party splenocytes added plates, indicating that third party CIK cells could have direct effects against highly activated T-cells.To further clarify the mechanisms of how third party CIK cells reduce the highly activated T-cells, we performed MLR as mentioned above with/without the neutralizing antibody against NKG2D, which is an activator receptor expressed on NK cells. Although CIK cells have cytotoxicity against tumor cells via NKG2D signaling pathway in part, adding NKG2D antibody did not show a statistical significant difference in MLR assays. It suggested that third party CIK cells could suppress alloreactive T-cells by several pathways other than NKG2D signaling pathway. Finally, we identified the in vivo kinetics of third party CIK cells after BMT. Third party derived CIK cells could be identified as a rare subpopulation in recipient peripheral blood for some days after CIK infusion, but all of them could not be detected anymore by day 7 after infusion. Whereas third party whole splenocytes expanded in recipient peripheral blood and mice received third party splenocytes with donor BM and splenocytes died until day7 because of GVHD induced by third party splenocytes. In conclusion, infusion of third party CIK cells has strong potential to prevent murine lethal GVHD probably due to selectively depleting highly proliferative alloreactive T-cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Dimethyl Fumarate Ameliorates Graft-Versus-Host Disease By Negatively Regulating Aerobic Glycolysis in Alloreactive T-Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 24-25
Author(s):  
Kiyomi Mashima ◽  
Kazuya Sato ◽  
Norihito Takayama ◽  
Junko Izawa ◽  
Takashi Ikeda ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Acute Gvhd ◽  
Aerobic Glycolysis ◽  
Dimethyl Fumarate ◽  
Activated T Cells ◽  
Graft Versus Host ◽  
Alloreactive T Cells ◽  
Glycolytic Activity

Background Dimethyl fumarate (DMF), a fumaric acid derivative, is currently used worldwide as a therapeutic agent for autoimmune diseases, such as multiple sclerosis and psoriasis. As an activator of Nrf-2, DMF protects cells from oxidative stress by inducing anti-oxidant enzymes. In addition, a recent report in Science has shown that DMF catalytically inactivates GAPDH, thereby reduces glycolytic activity, and results in immune modulation in activated CD4+ T-cells. We have previously shown that DMF and its metabolite monomethyl fumarate (MMF) significantly inhibit 3H-thymidine uptake in activated T-cells. DMF also decreased the expression of proliferation marker Ki-67 and intracellular IFN-γ of activated T-cells in a dose dependent manner. These findings prompted us to investigate whether DMF can be used for the treatment of graft-versus host disease (GVHD) after hematopoietic stem cell transplantation. In the current study, we investigated whether, and if so, how DMF inhibits human T-cell immune response and suppress acute GVHD in vivo using a xenogeneic GVHD mouse model. Methods To induce acute GVHD, human peripheral blood mononuclear cells (hPBMCs) were intravenously injected into sublethally irradiated (250 cGy) NOG mice. We allocated the mice into two groups; DMF treatment and non-treatment (control mice). Mice in the DMF group were administered DMF orally (100 mg/kg) for consecutive 7 days (day -3 to +3), and compared with the control mice treated with the same volume of vehicle. Results First, we observed that DMF treatment prolonged the survival of mice (Figure 1). Supporting the result, histopathological analysis showed that the number of hPBMCs infiltrated in the lungs and liver was decreased in the DMF group. Next, to identify the alteration of donor human cell populations after DMF treatment, hPBMCs were retrieved from the lungs on day 9 after transplantation and were analyzed by flow cytometry. Consistent with the histological findings, the absolute number of hPBMCs (hCD45+), and also T-cells (hCD45+hCD3+), in the lungs was significantly lower in the DMF group compared with the control (p &lt; 0.01) (Figure 2). Notably, the number of CD4+ T-cells, but not CD8+ T-cells, was decreased by the DMF treatment. The proportion of regulatory T-cells (Tregs) (hCD45+CD4+CD25+Foxp3+) was elevated in the DMF group, and this finding is consistent with existing reports that DMF may increase the proportion of Tregs. Furthermore, the expression level of PD-1 on hCD4+ T-cells was significantly lower in the DMF group. These results suggest that DMF treatment mainly regulates cell proliferation and functional differentiation of donor human CD4+ T-cells, leading to reduced severity of GVHD. Given that GAPDH and aerobic glycolysis have been shown as potential targets of DMF, we then measured glycolytic activity in human T-cells obtained from mice during GVHD. Extracellular acidification rate, an indicator of glycolytic activity, was monitored under basal conditions followed by sequential treatment with glucose, oligomycin, and 2-deoxy-D-glucose (a competitive inhibitor of glucose). Glycolytic activity after the addition of glucose was significantly lower in the T-cells of DMF group than in those of the control group (Figure 3). DMF treatment also led to a significant reduction in glycolytic capacity and glycolytic reserve. Furthermore, the oxygen consumption rate, an indicator of oxidative phosphorylation, was decreased in the DMF group, indicating that DMF disrupts mitochondrial energy production in T-cells, either directly or indirectly. Similar results were obtained from CD4+ T-cells. These results suggest that DMF treatment can negatively regulate aerobic glycolysis in alloreactive T-cells, leading to the mitigation of GVHD. Conclusion Oral administration of DMF ameliorates GVHD and prolongs the survival of mice by reducing donor CD4+ T-cell proliferation, while the number of Tregs is maintained. Our data suggests that DMF treatment drives donor T-cells into a metabolically inactive state by inhibiting aerobic glycolysis. This investigation provides pre-clinical data to use oral DMF as a prophylactic agent for acute GVHD. Disclosures Kanda: Daiichi Sankyo: Honoraria; Shire: Honoraria; Alexion Pharmaceuticals: Honoraria; Takeda Pharmaceuticals: Honoraria; Novartis: Honoraria; Kyowa Kirin: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria; Celgene: Honoraria; Otsuka: Honoraria, Research Funding; Chugai Pharma: Honoraria, Research Funding; Janssen: Honoraria; Astellas Pharma: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Merck Sharp & Dohme: Honoraria; Mochida Pharmaceutical: Honoraria; Mundipharma: Honoraria; Sanofi: Honoraria, Research Funding; Meiji Seika Kaisha: Honoraria; Bristol-Myers Squibb: Honoraria; Shionogi: Research Funding; Ono Pharmaceutical: Honoraria; Nippon Shinyaku: Honoraria, Research Funding.

Prevention of Graft-Versus-Host Disease by Selective Depletion of Alloreactive T Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3174-3174
Author(s):  
NgocDiep Le ◽  
Nelson Chao
Keyword(s):  
T Cells ◽  
Graft Versus Host Disease ◽  
Third Party ◽  
Dependent Manner ◽  
Activated T Cells ◽  
Host Disease ◽  
Graft Versus Host ◽  
Viral Antigens ◽  
Alloreactive T Cells ◽  
Donor T Cells

Abstract OBJECTIVE: Mismatched allogeneic hematopoietic cell transplantation (alloHCT) carries a high risk of life-threatening graft-versus-host disease (GVHD) due to activation of donor T cells by antigens present on host cells. Removal of donor mature T cells can prevent GVHD but leads to an increased incidence of opportunistic infections and disease relapse. This study aims to selectively deplete host-reactive donor T cells responsible for GVHD while preserving T cells with anti-tumor and anti-viral effects. METHODS: We utilized a photosensitizer, 4,5-dibromorhodamine-methyl ester (TH9402, Celmed Biosciences Inc., Saint-Laurent, Canada), in an ex vivo photodynamic purging (PDP) process to specifically eradicate host-reactive T cells. Donor T cells with anti-host specificity were identified in a unidirectional mixed lymphocyte culture (MLC) where they were activated and became proliferating. TH9402 is taken up by all cells and extruded out of the cell by P-glycoprotein (Pgp) in non-activated cells. However, due to inactivation of Pgp in activated T cells, TH9402 is retained in the mitochondria. Upon exposure to 514 nm light in the Theralux™ device (Celmed), it becomes extremely cytotoxic resulting in cell death. In this study, after treatment with various concentrations of TH9402, the cells were exposed to light for the elimination of alloreactive T cells. The efficiency of allodepletion was assessed by Granzyme B (GrB) assay. T-cell proliferation assays were used to demonstrate the preservation of anti-tumor and anti-viral effects. Finally, the skin explant assay, an in vitro model of GVHD, was utilized to examine the efficacy of the PDP treatment in the removal of alloreactive T cells responsible for GVHD. The parameters of the PDP treatment were optimized for use in subsequent clinical studies. RESULTS: After 72-hour MLC, optimal proliferative response was obtained at a responder: stimulator ratio of 1:1. Activated T cells expressed high level of activation markers such as CD25 and CD69. After the PDP treatment with 20μM of TH9402, alloreactive T cells were consistently depleted by more than 2 logs (Figure 1). Moreover, the PDP treatment did not significantly affect anti-tumor and anti-viral effects as evidenced by responses to third-party stimulators (Figure 2A), cytomegalovirus (CMV) (Figure 2B) and Candida antigens. Most importantly, co-culture of recipient’s skin with PDP-treated cells showed a reduction of graft-versus-host reaction (GVHR) in a TH9402-dose dependent manner. The PDP treatment with 20μM of TH9402 completely abolished GVHR in a skin explant assay. CONCLUSIONS: The PDP treatment can effectively remove donor T cells responsible for GVHD while preserve T cells with anti-tumor and anti-viral effects. These preclinical results provide a basis for initiating a clinical trial to assess the feasibility and efficacy of infusing PDP-treated donor T cells to alloHCT recipient in order to augment anti-tumor and anti-pathogen effects without causing GVHD. Figure 1 PDP treatment reduceds the frequency of alloreactive T cells in a TH9402 does dependent manner. Figure 1. PDP treatment reduceds the frequency of alloreactive T cells in a TH9402 does dependent manner. Figure 2 PDP treatment preserves responses to third-party stimulator and viral antigens. Figure 2. PDP treatment preserves responses to third-party stimulator and viral antigens.

A Functional Assay for Peripheral Blood T Cells to Predict Acute Graft-Versus-Host Disease.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4497-4497
Author(s):  
Carina A Baeuerlein ◽  
Christian Brede ◽  
Simone S Riedel ◽  
Ana-Laura Jordán-Garrote ◽  
Carolin Kiesel ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Functional Assay ◽  
Graft Versus Host ◽  
Initiation Phase ◽  
Alloreactive T Cells ◽  
Effector Phase ◽  
Donor T Cells

Abstract Abstract 4497 Patients undergoing allogeneic hematopoietic cell transplantation (allo-HCT) are at high risk to develop acute graft-versus-host disease (aGVHD) which is caused by alloreactive donor T cells. Early diagnosis of aGVHD remains difficult: there are no efficient methods to identify patients at risk of aGVHD, which could improve disease prevention. Therefore, to potentially predict aGVHD, we asked whether it is possible to detect in vivo activated alloreactive T cells in the peripheral blood immediately before entering aGVHD target tissues. To address this question, we used the CD107a/b degranulation assay (Betts et al., J Immunol Methods 2003 281:65) to measure cytotoxicity responses of alloractive T cells in a mouse model of aGVHD that allows us to trace alloreactive T cells in different phases of aGVHD by flow cytometry. Utilizing bioluminescence imaging in this model we have previously observed two distinct phases in aGVHD pathophysiology (Blood 2005;106:1113): During the initiation phase until day+3 alloreactive T cells are activated, proliferate in secondary lymphoid organs and acquire appropriate homing receptors to migrate into target tissues during the effector phase. In preparation for transplantation experiments, we tested T cell receptor transgenic (OT-1) T cells, which recognize the SIINFEKL-H-2b complex as a positive control. We observed degranulation of in vitro activated OT-1 T cells (84.40 ± 1.10%) against SIINFEKL-H-2b+ targets in contrast to C57Bl/6 wildtype targets (53.17 ± 2.65%). Unstimulated OT-1 T cells degranulated to a lower extend (22.93 ± 1.05%) against SIINFEKL-H-2b+ targets but less against SIINFEKL negative targets (9.47 ± 0.56%) (t-tests p<0.0001). We then transplanted 1,2×106 C57Bl/6 CD4+ and CD8+ T cells (H-2b, Thy1.1+) plus 5×106 C57Bl/6 bone marrow (BM) cells (H-2b, Thy1.2+) into myeloablative conditioned allogeneic Balb/c (H-2d, Thy1.2+, 8 Gy) recipients. As syngeneic controls we used C57Bl/6 (H-2b, Thy1.2+, 9 Gy) recipients. To prove specificity of the functional assay we transplanted 1,2×106 TCR transgenic OT-1 T cells (H-2b, Thy1.2+) plus 5×106 C57Bl/6 BM into conditioned C57Bl/6 (H-2b, Thy1.2+, 9 Gy) mice that expressed the SIINFEKL-H-2b+ complex ubiquitously. On day+2 and day+5 after HCT we analyzed the peripherial blood of the transplanted mice. As expected we did not detect donor T cells in the peripheral blood on day+2 during the GVHD initiation phase. However, by day+5 at the transition to the aGVHD effector phase large numbers of T cells had entered the circulation. Employing the degranulation assay revealed that peripheral blood CD8+ T cells from allogeneic recipients (C57Bl/6 □ Balb/c) degranulated against allogeneic targets (29.08 ± 4.46%), antigen specific CD8+ OT-1 T cells against SIINFEKL expressing targets (43.10 ± 3.78%) but less against SIINFEKL negative targets (9.78 ± 2.64%) (ANOVA p<0.0001). Reactive T cells in syngeneic controls were negligible (< 3%). Importantly, subsequent histopathological analysis of the same allogeneic recipients (where alloreactive T cells had degranulated) revealed aGVHD of the intestinal tract (grade 2-3) and the liver (0-2). No signs of GVHD were observed in mice where T cells had not degranulated (syngeneic controls). These preclinical data from our in vivo experiments encourage translation of this predictive test to patients undergoing allo-HCT. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Signaling Through the Type 2 Cannabinoid Receptor Regulates the Severity of Acute and Chronic Graft versus Host Disease

Blood ◽  
2020 ◽  
Author(s):  
Cheng Yin Yuan ◽  
Vivian Zhou ◽  
Garrett Sauber ◽  
Todd M Stollenwerk ◽  
Richard Komorowski ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Graft Versus Host Disease ◽  
Cd8 T Cells ◽  
Acute Gvhd ◽  
Cannabinoid Receptor ◽  
Therapeutic Targeting ◽  
Host Disease ◽  
Graft Versus Host

Graft versus host disease (GVHD) pathophysiology is a complex interplay between cells that comprise the adaptive and innate arms of the immune system. Effective prophylactic strategies are therefore contingent upon approaches that address contributions from both immune cell compartments. In the current study, we examined the role of the type 2 cannabinoid receptor (CB2R) which is expressed on nearly all immune cells and demonstrated that absence of the CB2R on donor CD4+ or CD8+ T cells, or administration of a selective CB2R pharmacological antagonist, exacerbated acute GVHD lethality. This was accompanied primarily by the expansion of proinflammatory CD8+ T cells indicating that constitutive CB2R expression on T cells preferentially regulated CD8+ T cell alloreactivity. Using a novel CB2R-EGFP reporter mouse, we observed significant loss of CB2R expression on T cells, but not macrophages, during acute GVHD, indicative of differential alterations in receptor expression under inflammatory conditions. Therapeutic targeting of the CB2R with the agonists, tetrahydrocannabinol (THC) and JWH-133, revealed that only THC mitigated lethal T cell-mediated acute GVHD. Conversely, only JWH-133 was effective in a sclerodermatous chronic GVHD model where macrophages contribute to disease biology. In vitro, both THC and JWH-133 induced arrestin recruitment and ERK phosphorylation via CB2R, but THC had no effect on CB2R-mediated inhibition of adenylyl cyclase. These studies demonstrate that the CB2R plays a critical role in the regulation of GVHD and suggest that effective therapeutic targeting is dependent upon agonist signaling characteristics and receptor selectivity in conjunction with the composition of pathogenic immune effector cells.

scholarly journals Graft-Versus-Host Disease–Free Antitumoral Signature After Allogeneic Donor Lymphocyte Injection Identified by Proteomics and Systems Biology

2019 ◽  
pp. 1-11 ◽  
Author(s):  
Xiaowen Liu ◽  
Zongliang Yue ◽  
Yimou Cao ◽  
Lauren Taylor ◽  
Qing Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
Systems Biology ◽  
Graft Versus Host Disease ◽  
Acute Gvhd ◽  
Hematopoietic Cell Transplantation ◽  
Training Set ◽  
Activated T Cells ◽  
Graft Versus Host ◽  
Single Cell Profiling ◽  
Validation Set

PURPOSE As a tumor immunotherapy, allogeneic hematopoietic cell transplantation with subsequent donor lymphocyte injection (DLI) aims to induce the graft-versus-tumor (GVT) effect but often also leads to acute graft-versus-host disease (GVHD). Plasma tests that can predict the likelihood of GVT without GVHD are still needed. PATIENTS AND METHODS We first used an intact-protein analysis system to profile the plasma proteome post-DLI of patients who experienced GVT and acute GVHD for comparison with the proteome of patients who experienced GVT without GVHD in a training set. Our novel six-step systems biology analysis involved removing common proteins and GVHD-specific proteins, creating a protein-protein interaction network, calculating relevance and penalty scores, and visualizing candidate biomarkers in gene networks. We then performed a second proteomics experiment in a validation set of patients who experienced GVT without acute GVHD after DLI for comparison with the proteome of patients before DLI. We next combined the two experiments to define a biologically relevant signature of GVT without GVHD. An independent experiment with single-cell profiling in tumor antigen–activated T cells from a patient with post–hematopoietic cell transplantation relapse was performed. RESULTS The approach provided a list of 46 proteins in the training set, and 30 proteins in the validation set were associated with GVT without GVHD. The combination of the two experiments defined a unique 61-protein signature of GVT without GVHD. Finally, the single-cell profiling in activated T cells found 43 of the 61 genes. Novel markers, such as RPL23, ILF2, CD58, and CRTAM, were identified and could be extended to other antitumoral responses. CONCLUSION Our multiomic analysis provides, to our knowledge, the first human plasma signature for GVT without GVHD. Risk stratification on the basis of this signature would allow for customized treatment plans.

Improvement of Selective Depletion of Alloreactive T Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1067-1067
Author(s):  
Erkut Bahceci ◽  
Marina Komarovskaya ◽  
Robert Kreitman ◽  
Dennis Cooper ◽  
Ira Pastan
Keyword(s):  
T Cells ◽  
Culture Media ◽  
Ex Vivo ◽  
Residual Activity ◽  
Third Party ◽  
Thymidine Uptake ◽  
Activated T Cells ◽  
Hematopoietic Stem ◽  
Alloreactive T Cells ◽  
Selective Depletion

Abstract Severe T cell depletion required for allogeneic hematopoietic stem cell transplantation from haplo-identical donors results in poor immune reconstitution and leads to high rates of mortality from infections, and relapse. One approach to overcome this problem is to infuse T cells depleted of alloreactivity. Selective depletion (SD) of alloreactive T cells is achieved by elimination of activated T cells after ex-vivo stimulation with recipient cells. To determine optimum selective depletion conditions, we have investigated the factors that modify alloreactivity of T cells. Methods: Alloreactivity was measured by one-way mixed lymphocyte reaction (MLR) using 3H thymidine uptake. PBMCs were used as responders and either irradiated expanded T cells (expT) or dendritic cells (DCs) as stimulators. T cells were expanded using anti-CD3 coated beads. DCs were generated from monocytes by GM-CSF and IL-4 stimulation. Selective depletion was performed by co-incubation of responder and stimulator cells for 72 hours and depletion of activated cells by an immunotoxin, LMB-2 (Anti-Tac (Fv)-PE-38), which was added to the culture at 24 and 48 hours. Effectiveness of the depletion was tested by a secondary MLR utilizing the original stimulator cells and unrelated third part cells. Results: Expansion of T cells has resulted in increase of HLA-DR, CD80 and CD86 expression compared to resting T cells (52.5% vs. 6%, 20.9% vs. 0.9%, and 32.9% vs. 20.9%, respectively), resulting in better stimulation in MLR (6505 cpm vs 1620 cpm). In one-way MLR using either PBMCs or CD25 depleted PBMCs as responders and expanded T cells and DCs as targets, with or without anti-CD28 in the culture media. DCs were better stimulators than expT cells (6636 vs. 4308). However, most dramatic effect was seen when anti-CD28 was added to the culture, increasing response to both expT cells and to a lesser extent DCs (40,169 and 19,303). Removal of CD25 positive cells also improved alloreactivity in all culture conditions (6636 in expT, 16,644 in DC, 57,363 in expT+CD28, and 30,943 in DC+CD28). To better define the effect of the target, we have performed Vbeta repertoire analysis of responding cells after expT cell, DC and expT cell+anti-CD28 stimulation. Flow cytometry revealed expansion of discrete Vbeta families, in addition to shared ones. We have then performed selective depletion using PBMCs or CD 25 depleted PBMCs as stimulators and expT cells, expT cell+anti-CD28, and DCs as stimulators. Residual alloreactivity after expT cell stimulation against original stimulators, DCs and third party cells were 7%, 147% and 99% respectively. Interestingly, after SD utilizing DCs as stimulators, there was substantial residual activity against expT cells (69%). When SD was performed using expT cells as stimulators with anti-CD28, combined with CD25 depletion, the depletion against both original stimulators and DCs was improved (2% and 54%, respectively). Conclusion: Depletion of regulatory T cells, and co-stimulation with ant-CD28 improves alloreactivity and selective depletion. Whether improvement in in-vitro selective depletion will result in better clinical outcome will be tested in a clinical trial.

The Role of Selective Depletion of Alloreactive Donor T Cells in Graft-Versus-Host Disease after Allogeneic Bone Marrow Transplantation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5167-5167
Author(s):  
Yihuan Chai ◽  
Huiying Qiu ◽  
Hui Lv
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Graft Versus Host Disease ◽  
Acute Gvhd ◽  
Allogeneic Bone Marrow ◽  
Third Party ◽  
Allogeneic Bone ◽  
Host Disease ◽  
Graft Versus Host ◽  
Donor T Cells

Abstract One of the main goals in allogeneic bone marrow(BM) transplantation is the abrogation of graft-versus-host disease (GVHD) with the preservation of antileukemia and antiviral activity. The Study present a selective T cell depletion strategy based on the physical separation of the alloreactive T cells, which were identified by expression of two activation-induced antigens (CD25 and CD69). T cells from C57BL/6(H-2b) mice were first activated with BALB/c (H-2d) recipient spleen cells in a 2-day mixed-lymphocyte-culture (MLC). Following this activation, this compound is selectively depleted based on expression of two activation-induced antigens CD25 and CD69 using magnetic cell sorting. The depleted cells or the untreated cells were then rechallenged respectively in a secondary MLC, with the same stimulator cells or a third-party (DBAH-2k) or tumor- specific (SP2/0, BALB/c-origin myeloma) cells. Cells proliferation were assayed at the indicated time points(1, 2, 3, 4, 5 days). These treated cells or control-cultured cells (2.0×106) mixed with 5.0×106 BM cells from C57BL/6 were transfused respectively by the trail vain into the lethally irradiated BALB/c to observe the survival time, GVHD incidence and pathological analysis. MLC assays demonstrated that this technique led to a significant decrease in alloreactivity of donor cells(29.02~64.17%), which at the same time preserved reactivity against third party cells(49.61~75.69%)and anti-tumor cells(61.14~68.62%). The mice in the group of control-coclutured were died of acute GVHD within 24days. The 7 recipient mice in the treated group were free of acute GVHD, and 3 mice were died of acute GVHD (aGVHD) within 23 days. MACS-based ex-vivo depletion of alloreactive donor T cells based on expression of two activation-induced antigens (CD25 and CD69) could inhibit anti-host responses, by contrast, anti-SP2/O and anti-third-party responses were preserved. Cotransplantation of these selected depleted cells and BM cells could reduce aGVHD.

Crosstalk Between the Bone and Immune Systems: Osteoclasts Function as Antigen-Presenting Cells and Activate CD4+ and CD8+ T Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 918-918
Author(s):  
Haiyan Li ◽  
Sungyoul Hong ◽  
Jianfei Qian ◽  
Jing Yang ◽  
Liang Zhang ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Multiple Myeloma ◽  
T Cells ◽  
Immune System ◽  
Mhc Class I ◽  
Cd8 T Cells ◽  
Immune Systems ◽  
Alloreactive T Cells ◽  
Antigen Presenting ◽  
Stimulation Of

Abstract Abstract 918 Bone is a dynamic tissue that is constantly being remodeled by bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OB). Bone destruction, observed in patients with autoimmune disease rheumatoid arthritis and malignancies such as multiple myeloma, is believed to be caused by hyperactivation of OCs. OCs differentiate from hematopoietic monocytic precursors under stimulation by the cytokines RANKL (receptor activator of nuclear factor κB ligand) and M-CSF (macrophage colony-stimulating factor), which are produced primarily by bone marrow stromal cells, OBs, and activated T cells. As OCs are derived from monocytes/macrophages lineage similar to dendritic cells (DCs), we hypothesized that OCs could serve as antigen-presenting cells (APCs) to activate T cells. In this study, OCs were generated from human monocytes with the stimulation of RANKL and M-CSF in vitro. DCs derived from monocytes with the stimulation of GM-CSF and IL-4 were used as positive controls. First, we examined the phenotype of OCs by flow cytometry, and OCs were detached by non-enzymatic cell dissociation solution. Results showed that OCs expressed MHC class I and II molecules and costimulatory molecules important for APCs such as CD80, CD86 and CD40. The expression of these molecules could be upregulated by LPS and IFN-γ. Second, we showed by PCR that OCs expressed IL-10, TGF-β, IL-6 and TNF-α, but not IL-12p35 and p40. Third, we examined the ability of OCs to present alloantigens and activate alloreactive T cells in a mixed lymphocyte reaction assay. OCs could present allogeneic antigens and activate both CD4+ and CD8+ alloreactive T cells to proliferate as detected by [3H]thymidine incorporation and CFSE dilution assay. The activation was restricted by MHC class I and II molecules. Finally, we recruited tetanus toxoid (TT)-immunized healthy donors to test whether OCs could uptake exogenous soluble antigens and present them to CD4+ T cells. OCs pulsed with TT could activate autologous specific CD4+ T cells, which was MHC II molecule restricted. These findings indicate that OCs could function as APCs and activate both CD4+ and CD8+ T cells. Thus, our study provides new insight to the effect of OCs on the immune system especially T cells. This is not only important for a better understanding of the crosstalk between the bone and immune system but also may help develop novel strategies for treating diseases such as rheumatoid arthritis and multiple myeloma, which affect both the bone and immune systems. Disclosures: No relevant conflicts of interest to declare.

Distinct roles for donor- and host-derived antigen-presenting cells and costimulatory molecules in murine chronic graft-versus-host disease: requirements depend on target organ

Blood ◽  
2005 ◽  
Vol 105 (5) ◽  
pp. 2227-2234 ◽  
Author(s):  
Britt E. Anderson ◽  
Jennifer M. McNiff ◽  
Dhanpat Jain ◽  
Bruce R. Blazar ◽  
Warren D. Shlomchik ◽  
...  
Keyword(s):  
Graft Versus Host Disease ◽  
Cd8 T Cells ◽  
Acute Gvhd ◽  
Dominant Role ◽  
Chronic Gvhd ◽  
Antigen Presenting Cells ◽  
Target Tissue ◽  
Host Disease ◽  
Graft Versus Host ◽  
Antigen Presenting

AbstractThe application of allogeneic stem cell transplantation (alloSCT) is limited by graft-versus-host disease (GVHD). GVHD can be divided into acute and chronic forms that likely have different requirements for initiation and pathogenesis mechanisms. In prior studies we demonstrated that residual host antigen-presenting cells (APCs) were required to initiate acute GVHD (aGVHD) mediated by CD8 T cells. In contrast, here we demonstrate that either donor or host APCs can initiate CD4-mediated GVHD in a model that has features of chronic GVHD (cGVHD). Both donor and host APCs must provide CD80/86-dependent costimulation to elicit maximal cGVHD, and there is no GVHD when both donor and host lack CD80/86. Finally, we were surprised to find that, although either donor or host APCs are sufficient to stimulate skin cGVHD, donor APCs play a dominant role in intestinal cGVHD. Both CD40 and CD80/86 are critical for donor APC function in intestinal cGVHD, but only CD80/86 is required for skin cGVHD. Thus, there are target-tissue–specific differences in APC requirements. These results identify differences in APC requirements between CD8-mediated aGVHD and CD4-mediated cGVHD. They further highlight donor APCs as additional targets for GVHD therapy.

P-glycoprotein targeting: a unique strategy to selectively eliminate immunoreactive T cells

Blood ◽  
2002 ◽  
Vol 100 (2) ◽  
pp. 375-382 ◽  
Author(s):  
Martin Guimond ◽  
Antonia Balassy ◽  
Mélanie Barrette ◽  
Sylvie Brochu ◽  
Claude Perreault ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Third Party ◽  
Cell Populations ◽  
Activated T Cells ◽  
Graft Versus Host ◽  
Histocompatibility Complex ◽  
P Glycoprotein ◽  
Selective Elimination

Abstract T lymphocytes have been found to harbor P-glycoprotein (Pgp) and to demonstrate modulation of its ion channel transporter function according to the state of activation of T lymphocytes. We hypothesized that cytotoxic chemicals that are extruded by Pgp could be used to specifically eliminate immunoreactive T-cell populations. In this study, we evaluated the capacity of 4,5-dibromorhodamine methyl ester (TH9402), a photosensitizer structurally similar to rhodamine, a dye transported by Pgp, and which becomes highly cytotoxic on activation with visible light to selectively deplete alloreactive T lymphocytes. Stimulation of T cells with mitogens or allogeneic major histocompatibility complex–mismatched cells resulted in the preferential retention of the TH9402 rhodamine-derivative in activated T cells, both CD4+ and CD8+. Photodynamic cell therapy of TH9402-exposed T cells led to the selective elimination of immunoreactive T-cell populations. In addition, this treatment preserved resting T cells and their capacity to respond to third-party cells. Inhibition of Pgp enhanced cellular trapping of the dye in nonactivated T cells and resulted in their depletion after exposure to light. Targeting of Pgp-deficient cells may therefore represent an appealing strategy for the prevention and treatment of graft-versus-host disease and other alloimmune or autoimmune disorders.

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