The infusion of ex vivo activated and expanded CD4+CD25+ immune regulatory cells inhibits graft-versus-host disease lethality

Blood ◽  
2002 ◽  
Vol 99 (10) ◽  
pp. 3493-3499 ◽  
Author(s):  
Patricia A. Taylor ◽  
Christopher J. Lees ◽  
Bruce R. Blazar
Keyword(s):  
T Cells ◽  
Graft Versus Host Disease ◽  
Cellular Therapy ◽  
Ex Vivo ◽  
Solid Organ Transplantation ◽  
Solid Organ ◽  
Host Disease ◽  
Graft Versus Host ◽  
In Vivo Animal Model

Immune regulatory CD4+CD25+ cells play a vital role in the induction and maintenance of self-tolerance and the prevention of autoimmunity. Recently, CD4+CD25+ cells have been shown to be required for the ex vivo induction of tolerance to alloantigen via costimulatory blockade and to inhibit allogeneic skin graft rejection. Data presented here demonstrate that CD4+CD25+ cells play an important role in graft-versus-host disease (GVHD) generation. Depletion of CD4+CD25+ cells from the donor T-cell inoculum or in vivo CD25-depletion of the recipient before transplantation resulted in increased GVHD mediated by CD4+or whole T cells in several strain combinations irrespective of the total body irradiation conditioning regime. The infusion of freshly purified donor CD4+CD25+ cells modestly inhibited GVHD when administered in equal numbers with whole CD4+ cells. Because CD4+CD25+ cells only account for 5% to 10% of the total CD4+ population, the administration of high numbers of fresh donor CD4+CD25+ cells may not be clinically practical. However, we found that large numbers of CD4+CD25+ cells can be obtained by ex vivo activation and expansion. Cultured CD4+CD25+ cells, administered in equal numbers with CD4+ T cells or CD25-depleted whole T cells, resulted in significant inhibition of rapidly lethal GVHD. To our knowledge, this study is the first to demonstrate that activated, cultured CD4+CD25+ cells can offer substantial protection in a relevant in vivo animal model of disease. These data have important ramifications for clinical bone marrow and solid organ transplantation. CD4+CD25+ cells warrant consideration as an exciting new modality of cellular therapy for the inhibition of undesirable autologous and allogeneic responses.

Adoptive Transfer Of Ex Vivo “Educated” CD4+CD25+FoxP3+ Regulatory T Cells Effectively Treats Acute Graft Versus Host Disease Preserving Graft Versus Tumor Effect

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4485-4485
Author(s):  
Antonio Pierini ◽  
Dominik Schneidawind ◽  
Mareike Florek ◽  
Maite Alvarez ◽  
Yuqiong Pan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Tumor Cells ◽  
Graft Versus Host Disease ◽  
Adoptive Transfer ◽  
Cellular Therapy ◽  
Ex Vivo ◽  
Host Disease ◽  
Graft Versus Host

Donor derived regulatory T cells (Tregs) effectively prevent graft versus host disease (GVHD) in mouse models and in early phase clinical trials. Interleukin 2 (IL-2) therapy in patients with chronic GVHD (cGVHD) can increase Treg number and the Treg/CD4+ T cell ratio resulting in organ damage reduction and symptom relief. Less is known regarding Treg-based treatment for acute GVHD (aGVHD). In this study we evaluated the role of donor Treg cellular therapy for aGVHD treatment in well established murine models. T cell depleted bone marrow (TCD BM) from C57BL/6 mice was transplanted into lethally irradiated (8 Gy) BALB/C recipients together with 7.5x105 to 1x106/animal donor derived luc+ Tcons. Naturally occurring CD4+CD25+FoxP3+ donor type Tregs (nTregs) were purified from C57BL/6 donor mice. 2.5x105/mouse nTregs were injected at day 6 or 7 after transplant in mice that showed clear clinical signs of aGVHD and Tcon proliferation assessed by bioluminescence imaging (BLI). Survival analysis showed a favorable trend for nTreg treated mice, but the impact of this treatment was modest and not statistically significant (p 0.08). aGVHD is a disease characterized by the activation and rapid proliferation of alloreactive donor conventional T cells (Tcons) directed against host antigens, so one of the major obstacles of this approach is to overcome the large number and effector function of activated Tcons. Several studies have utilized ex vivo expansion of Tregs to increase their number with the goal of maintaining suppressive function. We developed a different strategy with the intent to “educate” Tregs to specifically suppress the reactive Tcon population. We incubated 2.5x105 donor derived Tregs with irradiated (3000 cGy) blood of aGVHD affected mice for 20 hours without further stimulation and injected the entire pool of these cells, termed educated Treg (eTregs), at day 7 or 8 after transplant and Tcon injection. Interestingly eTregs significantly improved aGVHD affected mouse survival (p = 0.0025 vs Tcons alone). BLI showed no difference between the groups (p = 0.85) because the treatment intervened after Tcon proliferation and activation was initiated. To evaluate eTreg impact on graft versus tumor (GVT) effects, we transplanted BALB/C mice with C57BL/6 TCD BM and 1x104/mouse luc+ A20 tumor cells along with 1x106/mouse donor Tcons and 2.5x105 eTregs. Mice that received TCD BM and A20 tumor cells alone died from progressive tumor growth, while mice that received Tcons died from GVHD without tumor engraftment. Further animals that received both Tcon and eTreg treatment did not have tumor engraftment demonstrating that eTregs do not impact Tcon mediated GVT effects. Further studies are ongoing to characterize the eTreg population as compared to nTreg, with respect to expression of activation markers and in functional assays. Our observations indicate that Tregs can be ex vivo educated to suppress in vivo reactive and proliferating Tcons. Moreover our data demonstrate that eTreg adoptive transfer is clinically feasible and promising. These findings may be relevant for the development of clinical grade Treg based cellular therapy for the treatment of conditions caused by immune dysregulation such as aGVHD and autoimmune diseases and for transplant tolerance induction. Disclosures: No relevant conflicts of interest to declare.

Prevention of graft-versus-host-disease with preserved graft-versus-leukemia-effect by ex vivo and in vivo modulation of CD4+ T-cells

2013 ◽  
Vol 71 (11) ◽  
pp. 2135-2148 ◽  
Author(s):  
Stephan Fricke ◽  
Nadja Hilger ◽  
Christian Fricke ◽  
Uta Schönfelder ◽  
Gerhard Behre ◽  
...  
Keyword(s):  
T Cells ◽  
Graft Versus Host Disease ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Host Disease ◽  
Graft Versus Host ◽  
Graft Versus Leukemia ◽  
Graft Versus Leukemia Effect

scholarly journals Attenuation of graft-versus-host disease and graft rejection by ex vivo immunotoxin elimination of alloreactive T cells in an H-2 haplotype disparate mouse combination

Blood ◽  
1994 ◽  
Vol 83 (1) ◽  
pp. 288-298 ◽  
Author(s):  
M Cavazzana-Calvo ◽  
JL Stephan ◽  
S Sarnacki ◽  
S Chevret ◽  
C Fromont ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Graft Versus Host Disease ◽  
Graft Rejection ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
In Vivo Model ◽  
Host Disease ◽  
Graft Versus Host

A mouse anti-interleukin-2 receptor A-chain-specific PC61-immunotoxin (PC61-IT) strongly inhibited a primary mixed lymphocyte culture and major histocompatibility complex (MHC)-restricted cytotoxicity. The allodepleted T cells retained their proliferative and cytotoxic capacities in response to third-party stimulation, showing that PC61-IT specifically deleted recipient antigen-specific T-cell clones from the donor mouse. The ability of this specific allodepletion to prevent graft-versus-host disease (GVHD) and graft rejection was investigated in vivo. IT-depleted, activated parental T lymphocytes (C3H/eB) were intravenously injected into lethally irradiated CDF1 mice. GVHD was evaluated after 6 days on the severity of gut lesions. PC61-IT-treated cells significantly reduced both donor T-cell infiltration and acceleration of epithelial renewal (a sensitive index of gut damage) as compared with those for the corresponding untreated controls. The effect of selective allo-depletion on prevention of GVHD and graft rejection was further studied after MHC-haploincompatible bone marrow (BM) transplantation. A significant increase in survival was observed in mice receiving 2 x 10(6) T-cell-depleted BM cells and 0.5 x 10(6) PC61-IT-treated T cells, because one-third were alive without GVHD (and with stable full or partial engraftment) after 100 days, whereas all the mice infused with BM and sham-treated T cells died within 80 days from GVHD, and all the mice infused with BM cells alone rejected grafts. Furthermore, specific tolerance in chimeras towards donor cells could be shown. These results as observed in an experimental in vivo model corroborate previous results obtained in vitro in humans and lead us to consider the use of this selective allodepletion in human BM transplant from donors other than identical familial siblings.

scholarly journals A Selective TNFR2 Agonist Expands Host Treg Cells in Vivo to Protect from Acute Graft-Versus-Host Disease

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1099-1099 ◽  
Author(s):  
Andreas Beilhack ◽  
Martin Chopra ◽  
Marlene Biehl ◽  
Martin Vaeth ◽  
Andreas Brandl ◽  
...  
Keyword(s):  
T Cells ◽  
Graft Versus Host Disease ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Peripheral Tissues ◽  
Host Disease ◽  
Graft Versus Host ◽  
Pre Treatment

Abstract Donor CD4+Foxp3+ regulatory T cells (Tregs) suppress graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HCT) while maintaining the anti-tumoral effect of transplanted conventional T cells in preclinical mouse models. Current clinical study protocols with donor Tregs for treatment or prophylaxis of GVHD rely on their ex vivo expansion and infusion in high numbers. Here we present a fundamentally novel strategy for inhibiting GVHD that is based on the in vivo expansion of recipient Tregs prior to allo-HCT, exploiting the crucial role of tumor necrosis factor receptor 2 (TNFR2) in Treg biology. To this end we constructed a recombinant nonameric TNFR2-specific agonist, mimicking the activity of murine membrane-bound TNF on TNFR2 without TNFR1 stimulation, thereby avoiding the inflammatory side effects observed with conventional TNF. In vitro, this TNFR2-agonist expanded natural Tregs from wild type but not from TNFR2 KO mice. Accordingly, a human variant of this TNFR2-specific agonist expanded human Tregsin vitro. In vivo treatment of healthy mice with the murine TNFR2-agonist significantly increased Treg numbers in secondary lymphoid organs and peripheral tissues, particularly in the gastrointestinal tract, a prime target of acute GVHD. Next, we pre-treated recipient mice with this novel TNFR2-agonist to expand host-type radiation resistant Tregs prior to of allo-HCT in two models across MHC barriers (C57BL/6, H-2b->Balb/c, H-2d and FVB/N, H-2q->C57BL/6, H-2b). TNFR2-agonist pre-treatment resulted in significantly prolonged survival and reduced GVHD severity when compared to TNFR2-deficient recipients or untreated allo-HCT recipients. This was accompanied by reduced donor T cell proliferation and infiltration into GVHD target organs as assessed by in vivo and ex vivo bioluminescence imaging, flow cytometry and immunofluorescence microscopy. While in vivo TNFR2-agonist pre-treatment protected allo-HCT recipients from GVHD, anti-tumor effects of transplanted T cells remained unaffected in two different murine B cell leukemia models. In vivo depletion of host derived Tregs completely abrogated the protective effect of TNFR2-agonist pre-treatment. Our study shows that the expansion of host Tregs by selective in vivo TNFR2-activation significantly improves the outcome after allo-HCT and results in prolonged tumor-free survival. Disclosures No relevant conflicts of interest to declare.

scholarly journals Ex vivo fucosylation of third-party human regulatory T cells enhances anti–graft-versus-host disease potency in vivo

Blood ◽  
2015 ◽  
Vol 125 (9) ◽  
pp. 1502-1506 ◽  
Author(s):  
Simrit Parmar ◽  
Xiaoying Liu ◽  
Amer Najjar ◽  
Nina Shah ◽  
Hong Yang ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Graft Versus Host Disease ◽  
Ex Vivo ◽  
Third Party ◽  
Host Disease ◽  
Graft Versus Host ◽  
Cell Dose ◽  
Key Points

Key Points Fucosylated Tregs persist for a longer time in vivo. Fucosylated Tregs are able to prevent GVHD at a lower cell dose compared with untreated Tregs.

scholarly journals Attenuation of graft-versus-host disease and graft rejection by ex vivo immunotoxin elimination of alloreactive T cells in an H-2 haplotype disparate mouse combination

Blood ◽  
1994 ◽  
Vol 83 (1) ◽  
pp. 288-298 ◽  
Author(s):  
M Cavazzana-Calvo ◽  
JL Stephan ◽  
S Sarnacki ◽  
S Chevret ◽  
C Fromont ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Graft Versus Host Disease ◽  
Graft Rejection ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
In Vivo Model ◽  
Host Disease ◽  
Graft Versus Host

Abstract A mouse anti-interleukin-2 receptor A-chain-specific PC61-immunotoxin (PC61-IT) strongly inhibited a primary mixed lymphocyte culture and major histocompatibility complex (MHC)-restricted cytotoxicity. The allodepleted T cells retained their proliferative and cytotoxic capacities in response to third-party stimulation, showing that PC61-IT specifically deleted recipient antigen-specific T-cell clones from the donor mouse. The ability of this specific allodepletion to prevent graft-versus-host disease (GVHD) and graft rejection was investigated in vivo. IT-depleted, activated parental T lymphocytes (C3H/eB) were intravenously injected into lethally irradiated CDF1 mice. GVHD was evaluated after 6 days on the severity of gut lesions. PC61-IT-treated cells significantly reduced both donor T-cell infiltration and acceleration of epithelial renewal (a sensitive index of gut damage) as compared with those for the corresponding untreated controls. The effect of selective allo-depletion on prevention of GVHD and graft rejection was further studied after MHC-haploincompatible bone marrow (BM) transplantation. A significant increase in survival was observed in mice receiving 2 x 10(6) T-cell-depleted BM cells and 0.5 x 10(6) PC61-IT-treated T cells, because one-third were alive without GVHD (and with stable full or partial engraftment) after 100 days, whereas all the mice infused with BM and sham-treated T cells died within 80 days from GVHD, and all the mice infused with BM cells alone rejected grafts. Furthermore, specific tolerance in chimeras towards donor cells could be shown. These results as observed in an experimental in vivo model corroborate previous results obtained in vitro in humans and lead us to consider the use of this selective allodepletion in human BM transplant from donors other than identical familial siblings.

Anti-Murine Thymocyte Globulin Prevents Acute Graft-Versus-Host Disease and Induces Foxp3+ T Cells in Target Organs.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3251-3251 ◽  
Author(s):  
Melanie C. Ruzek ◽  
James S. Waire ◽  
William Weber ◽  
John M. Williams ◽  
Susan M. Richards ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Graft Versus Host Disease ◽  
Acute Gvhd ◽  
Solid Organ ◽  
Protective Effects ◽  
Host Disease ◽  
Graft Versus Host ◽  
Murine Thymocyte

Abstract Rabbit anti-human thymocyte globulins (ATG) (Thymoglobulin®) are widely used for treatment and prevention of solid organ transplant rejection. However, more recently these therapies have been shown to also reduce the severity of graft-versus-host disease (GVHD) following allogeneic stem cell transplantation. However, dose and schedule of ATG administration in the clinic has not been well defined, and thus, the studies reported here were undertaken to explore optimal dosing and timing regimens as well as possible mechanisms of action of anti-thymocyte globulin in an in vivo murine model of GVHD. We demonstrate that a murine version of ATG, rabbit anti-murine thymocyte globulin (mATG), completely inhibits the development of acute GVHD in a model of allosplenocyte transfer into immunodeficient recipient mice (C57BL/6→BALB/c RAG-2−/ −). This protection is observed even when mATG administration is delayed for up to three days following allosplenocyte transfer. Administration of mATG six or more days following induction of GVHD still ameliorates disease in up to 50% of the animals, depending on the timing of mATG treatment. Murine ATG also remains completely protective down to doses of 1mg/kg if administered at the time of allosplenocyte transfer. Although T cell depletion is still observed at this low, but efficacious dose of mATG, we also find significant increases in Foxp3+ CD4+ regulatory T cells in the spleen (30 fold over control) as well as increased Foxp3+ expression in liver and intestines (3 fold over control). These results demonstrate a potent protective effect of murine ATG in this model of acute GVHD and suggest that the induction of regulatory T cells may participate in the protective effects observed.

Fatty Acid Metabolism Provides a Potential Therapeutic Target To Treat Graft-Versus-Host Disease

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2002-2002
Author(s):  
Craig A. Byersdorfer ◽  
Victor Tkachev ◽  
Stefanie Goodell ◽  
Jacob Swanson ◽  
James L.M. Ferrara
Keyword(s):  
T Cells ◽  
Fatty Acid ◽  
Graft Versus Host Disease ◽  
Ex Vivo ◽  
Cell Metabolism ◽  
Cell Types ◽  
Host Disease ◽  
Graft Versus Host ◽  
Donor T Cells

Abstract The metabolism of lymphocytes activated in vivo remains poorly understood. Previous work has demonstrated that T cells activated during graft-versus-host disease (GVHD) adopt metabolic profiles distinct from other cell types. We hypothesized that a deeper understanding of allogeneic T cell metabolism, followed by exploitation of metabolic differences, might allow selective elimination of pathogenic T cells while preserving normal immune responses following bone marrow transplantation (BMT). We tested this hypothesis by evaluating fatty acid (FA) metabolism in proliferating donor T cells during a parent into F1 model of GVHD (C57Bl/6 into B6D2F1). Compared to naive donor T cells, T cells from allogeneic recipients increased FA transport (44.3 ±6.9% vs. 0.7 ±0.1%, p=0.003) and up-regulated a regulator of oxidative metabolism, PGC-1α (0.9 ±0.2 vs. 0.03 ±0.01, p=0.005). These changes were present in T cells recovered from both the liver and the spleen. Allogeneic T cells also up-regulated mRNA and protein levels for FA oxidation enzymes (e.g. CPT1a) and oxidized more fatty acids ex vivo. We confirmed these changes in a second, minor histocompatibility model of GVHD, where we observed significantly increased levels of FA transport, PGC-1α, and FA oxidation enzymes. To identify potential therapeutic targets selective for alloreactive T cells, we compared the metabolic profile of allogeneic T cells to the profile observed in T cells following acute activation in vivo. We injected OT-I T cells into naïve C57Bl/6 mice, challenged mice one day later with ovalbumin-bearing dendritic cells, and harvested T cells 7 days after immunization. OT-I T cells proliferated robustly to cellular immunization, but did not increase FA transport or levels of PGC-1α (Figure 1A,B). We then tested the ability of inhibitors of FA oxidation (targeting CPT1a) to selectively eliminate allogeneic T cells during GVHD. A single dose of etomoxir decreased the total number of donor T cells in allogeneic recipients by 35% and eight doses over 14 days significantly improved clinical GVHD scores (Figure 1C). Treatment with a second CPT1a inhibitor improved post-transplant survival (78% vs. 50%, inhibitor vs. PBS respectively). Importantly, CPT1a inhibition did not diminish the number of regulatory T cells or the number of T cells reconstituting via homeostatic proliferation following syngeneic BMT. In total, these data demonstrate that allogeneic T cells increase FA metabolism during GVHD and that this phenotype differentiates allogeneic cells from T cells responding to acute activation or proliferating during homeostatic reconstitution. This study challenges the paradigm of effector lymphocyte metabolism in vivo and we conclude that inhibition of FA oxidation may be a selective way to eliminate pathogenic T cells causing immune-mediated disease. Disclosures: Ferrara: University of Michigan: Patent for GVHD Biomarkers, Patent for GVHD Biomarkers Patents & Royalties.

Prevention of Transfusion-Associated Graft-Versus-Host Disease by Photochemical Treatment

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 3140-3147 ◽  
Author(s):  
Joshua A. Grass ◽  
Tamim Wafa ◽  
Aaron Reames ◽  
David Wages ◽  
Laurence Corash ◽  
...  
Keyword(s):  
T Cells ◽  
Gamma Radiation ◽  
Graft Versus Host Disease ◽  
Clinical Signs ◽  
Peripheral Blood Cell ◽  
Control Group ◽  
Host Disease ◽  
Graft Versus Host

Abstract Photochemical treatment (PCT) with the psoralen S-59 and long wavelength ultraviolet light (UVA) inactivates high titers of contaminating viruses, bacteria, and leukocytes in human platelet concentrates. The present study evaluated the efficacy of PCT to prevent transfusion-associated graft-versus-host disease (TA-GVHD) in vivo using a well-characterized parent to F1 murine transfusion model. Recipient mice in four treatment groups were transfused with 108 splenic leukocytes. (1) Control group mice received syngeneic splenic leukocyte transfusions; (2) GVHD group mice received untreated allogeneic splenic leukocytes; (3) gamma radiation group mice received gamma irradiated (2,500 cGy) allogeneic splenic leukocytes; and (4) PCT group mice received allogeneic splenic leukocytes treated with 150 μmol/L S-59 and 2.1 J/cm2UVA. Multiple biological and clinical parameters were used to monitor the development of TA-GVHD in recipient mice over a 10-week posttransfusion observation period: peripheral blood cell levels, spleen size, engraftment by donor T cells, thymic cellularity, clinical signs of TA-GVHD (weight loss, activity, posture, fur texture, skin integrity), and histologic lesions of liver, spleen, bone marrow, and skin. Mice in the control group remained healthy and free of detectable disease. Mice in the GVHD group developed clinical and histological lesions of TA-GVHD, including pancytopenia, marked splenomegaly, wasting, engraftment with donor derived T cells, and thymic hypoplasia. In contrast, mice transfused with splenic leukocytes treated with (2,500 cGy) gamma radiation or 150 μmol/L S-59 and 2.1 J/cm2 UVA remained healthy and did not develop detectable TA-GVHD. Using an in vitro T-cell proliferation assay, greater than 105.1 murine T cells were inactivated by PCT. Therefore, in addition to inactivating high levels of pathogenic viruses and bacteria in PC, these data indicate that PCT is an effective alternative to gamma irradiation for prevention of TA-GVHD.

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