Pre-Clinical Trial to Evaluate the Efficacy of Delayed Administration of Ixazomib in the Prophylaxis of Chronic Graft-Versus-Host Disease

Abstract Introduction: Although survival rates have improved over the years, chronic graft-versus-host disease (cGvHD) remains the most frequent and severe complication in the long term after allogeneic hematopoietic stem cell transplantation (allo-HSCT). All the strategies developed to reduce its incidence are based on procedures aimed to decrease the risk of acute GvHD that, consequently, can also reduce the risk of cGvHD, mainly using immunosuppression in the early post-transplant period. These strategies induce apoptosis of donor T lymphocytes, responsible for a/cGvHD but also for graft-versus-tumor response. In the present project, we have evaluated a new strategy aimed to reduce the risk of cGvHD by manipulating the immune response not in the early post-transplant period, but in later phases. cGvHD develops via a complex cellular and molecular network involving thymus damage and unusual antigen presentation leading to aberrant T- and B-cell reactions characterized by Th17/Tc17 differentiation, macrophage sequestration in tissue, alloantibody formation, and fibrosis. Most of these cell populations are dependent on NF-kB for their activation. Ixazomib is a second-generation proteasome inhibitor for oral administration, representing an ideal candidate for prophylaxis in GvHD. Objective: We propose to develop a murine model of cGVHD and progressive onset cGvHD to test the efficacy of delayed administration of ixazomib as a novel strategy to decrease the risk of cGvHD. Methods: For in vitro studies, peripheral blood mononuclear cells from healthy donors were stimulated in the presence of different concentrations of ixazomib. After 48h and 120h, apoptosis was analyzed, and activation markers were evaluated. For in vivo studies, a murine model of progressive cGvHD (pcGvHD - allowing the recipient to survive to mild aGvHD, thus favoring autoreactive T cells to expand and cause cGvHD) and a scleroderma model of cGvHD were used. Ixazomib at 0.75mg/Kg/twice weekly, 2X from day +21 in the pcGvHD, with or without cyclosporine A (CyA) at 5mg/Kg/day from day 0 until the end of the study. For scleroderma cGvHD group, 3mg/Kg/2X from day +30 was used up to 120 days post-transplant. Flow cytometry was used to evaluate the different lymphocyte populations in the different target organs of the GvHD. Results: In vitro results showed that activated T lymphocytes are sensitive to the proapoptotic effect of ixazomib (>100nM), while high concentrations of the drug are necessary to cause apoptosis in the non-activated cells (5000nM). Through CD27 and CD45 expression, we verified that ixazomib has a proapoptotic effect mainly on naïve and effector cells. We also verified a decrease in the activation markers of CD3+CD25+INF-γ+ cells (p˂0.01, 1000nM, n = 4). In the animal trial, the survival of the pcGvHD model mice treated with ixazomib was significantly higher as compared to untreated mice, with a significant decrease in the signs of pcGvHD (Figure 1A). In addition, the combination of CyA and ixazomib improved survival as compared to those mice receiving CyA or ixazomib alone as well as untreated controls (Figure 1B). In the scleroderma cGvHD model, we observed that the animals treated with ixazomib showed significantly less signs of GvHD (p˂0.0001) (Figure 2). Multiparametric flow cytometer analyses showed that in the pcGvHD model, mice treated with ixazomib had a decrease of effector CD4 T-cells in bone marrow (p=0.06) and spleen (p=0.015) when compared to untreated mice. Also an increase of CD19+ cells in the colon (p=0.04), liver (p=0.035), lymph nodes (LN) (p=0.037) and lungs (p=0.03) was observed. Regarding the regulatory T cells (Foxp3+), the treated mice had a significant increase in LN (p=0.02), Peyer patches (p=0.015) and thymus (p=0.028) as compared to untreated mice. Conclusion: In vitro studies show that ixazomib induces apoptosis on activated T lymphocytes and decreases the expression of activation markers. Our in vivo model indicates that the combination of CyA and ixazomib for the prevention of pcGvHD and cGVHD after allogeneic HSCT is promising and merits further investigation in clinical trials. Figure 1. Survival of pcGvHD. Figure 2. Score graphic of scleroderma cGvHD. BM - Bone marrow, mice that were transplanted with BM from BALBc mice (Syngeneic). Disclosures Ramos: Takeda Oncology: Research Funding.

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

Blood ◽

10.1182/blood.v93.9.3140 ◽

1999 ◽

Vol 93 (9) ◽

pp. 3140-3147 ◽

Cited By ~ 100

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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Pretreatment of murine donor grafts with L-leucyl-L-leucine methyl ester: elimination of graft-versus-host disease without detrimental effects on engraftment

Blood ◽

10.1182/blood.v75.3.798.798 ◽

1990 ◽

Vol 75 (3) ◽

pp. 798-805 ◽

Cited By ~ 14

Author(s):

BR Blazar ◽

DL Thiele ◽

DA Vallera

Keyword(s):

Bone Marrow ◽

T Cell ◽

Methyl Ester ◽

Graft Versus Host Disease ◽

Host Cells ◽

Host Disease ◽

Murine Bone Marrow ◽

Graft Versus Host

Abstract Incubation of murine bone marrow and splenocytes with the dipeptide methyl ester, L-leucyl-L-leucine methyl ester (Leu-Leu-OMe), which results in the selective depletion of cytotoxic T cells and their precursors, natural killer cells, and monocytes, completely protected 30 recipients of fully allogeneic donor grafts from lethal graft-versus- host disease (GVHD). These results were comparable with those obtained in 30 recipients of anti-Thy 1.2 plus complement (C')-treated donor marrow. However, in contrast to antibody- and C'-dependent T-cell depletion, which reduces the level of donor cell engraftment in our model system, we did not observe such effects using Leu-Leu-OMe marrow pretreatment. As compared with the 24 H-2 typed recipients of anti-Thy 1.2 + C'-treated donor grafts, the 29 H-2 typed recipients of Leu-Leu- OMe-treated donor grafts had significantly (P less than .001) higher percentages of donor cells (mean = 93% v 74%) and significantly (P less than .001) lower percentages of host cells (mean = 6% v 15%) posttransplantation. In vitro limiting dilution assay (LDA) was performed to assess the comparative efficacy of cytolytic T-lymphocyte (CTL) precursor depletion by Leu-Leu-OMe or anti-Thy 1.2 + C' pretreatment. We observed greater levels of CTL precursor depletion in Leu-Leu-OMe treated as compared with anti-Thy 1.2 + C'-treated bone marrow plus spleen cells (BMS) obtained from nontransplanted mice. This suggests that the in vivo results cannot simply be attributed to a less efficacious functional inactivation of cytolytic T-cell precursors by Leu-Leu-OMe treatment as compared with anti-Thy 1.2 + C' treatment. Immunoreconstitution was similar in recipients of Leu-Leu-OMe-treated grafts and anti-Thy 1.2 + C'-treated grafts 100 days posttransplant. In our opinion, Leu-Leu-OMe marrow pretreatment deserves further investigation as a methodology to achieve GVHD prevention without significantly reducing the propensity toward host cell repopulation.

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Characterization of in vitro antimurine thymocyte globulin–induced regulatory T cells that inhibit graft-versus-host disease in vivo

Blood ◽

10.1182/blood-2007-08-106526 ◽

2008 ◽

Vol 111 (3) ◽

pp. 1726-1734 ◽

Cited By ~ 36

Author(s):

Melanie C. Ruzek ◽

James S. Waire ◽

Deborah Hopkins ◽

Gina LaCorcia ◽

Jennifer Sullivan ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Graft Versus Host Disease ◽

Antithymocyte Globulin ◽

Mrna Level ◽

Regulatory Cells ◽

Host Disease ◽

Graft Versus Host

Abstract Antithymocyte/antilymphocyte globulins are polyclonal antihuman T-cell antibodies used clinically to treat acute transplant rejection. These reagents deplete T cells, but a rabbit antihuman thymocyte globulin has also been shown to induce regulatory T cells in vitro. To examine whether antithymocyte globulin–induced regulatory cells might be functional in vivo, we generated a corresponding rabbit antimurine thymocyte globulin (mATG) and tested its ability to induce regulatory cells in vitro and whether those cells can inhibit acute graft-versus-host disease (GVHD) in vivo upon adoptive transfer. In vitro, mATG induces a population of CD4+CD25+ T cells that express several cell surface molecules representative of regulatory T cells. These cells do not express Foxp3 at either the protein or mRNA level, but do show suppressive function both in vitro and in vivo when adoptively transferred into a model of GVHD. These results demonstrate that in a murine system, antithymocyte globulin induces cells with suppressive activity that also function in vivo to protect against acute GVHD. Thus, in both murine and human systems, antithymocyte globulins not only deplete T cells, but also appear to generate regulatory cells. The in vitro generation of regulatory cells by anti-thymocyte globulins could provide ad-ditional therapeutic modalities for immune-mediated disease.

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Host γd T cells Exacerbate Experimental Acute Graft-Versus-Host Disease through Activation of Host Antigen Presenting Cells.

Blood ◽

10.1182/blood.v104.11.3045.3045 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3045-3045

Author(s):

Yoshinobu Maeda ◽

Pavan Reddy ◽

Chen Liu ◽

D. Keith Bishop ◽

James L.M. Ferrara

Keyword(s):

T Cells ◽

T Cell ◽

Mhc Class Ii ◽

Graft Versus Host Disease ◽

Serum Levels ◽

Host Disease ◽

Graft Versus Host ◽

Full Intensity

Abstract Large numbers of T cells bearing γd T cell receptors are present in graft-versus-host disease (GVHD) target tissues. We investigated the potential role of host γd T cells during acute GVHD in a well-characterized GVHD model following full intensity conditioning (11 Gy TBI). BM and spleen T cells from BALB/c (H2d) donors were transplanted into wild type (wt) B6, aß T cell deficient B6 (aß −/−) or γd T cell deficient B6 (γd −/−) hosts. γd −/− hosts demonstrated significantly better day 35 survival (85%) than wt (40%) or aß−/− hosts (18%) (P<0.05). Reconstitution of γd −/− B6 hosts with B6 type γd T cells 24 hr prior to BMT restored lethal GVHD (50 % day 35 survival). In vivo, γd −/− B6 hosts demonstrated at least a five fold reduction in donor T cell expansion and cytokine production. In vitro, T cells proliferated less when co-cultured with allogeneic γd −/− dendritic cells (DCs) than with wt DCs (40,127 ± 1634 vs. 72,503 ± 1296, P<0.05). BM-derived DCs cultured with γd T cells caused greater proliferation of allogeneic T cells than DCs cultured with aß T cells (15.1 ± 21 x 104 vs. 5.1 ± 1.2 x 104, P<0.05). We next tested the effect of γd T cells on host DCs in vivo using a model system in which only the DCs injected prior to BMT expressed the alloantigen that stimulated the GVHD reaction. MHC Class II −/− B6 mice that had been depleted of γd T cells were given 11 Gy TBI and injected one day prior to BMT with B6 DCs that had been co-cultured either with γd T cells or with medium. On day 0 both groups of recipient mice were injected with BM plus splenic T cells from allogeneic bm12 donors. On day +5, CD4+ donor T cells expanded four times more in recipients of DCs co-cultured with γd T cells than in recipients of control DCs and serum levels of TNF-a were significantly higher (36.7 + 6.8 vs. 21.3 + 3.7 pg/ml, P<0.05). Together these data demonstrate that γd T cells amplify the stimulatory function of host DCs and increase the severity of GVHD, suggesting that a new therapeutic target for the prevention of the major BMT toxicity.

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Donor Dual TCR T Cells Preferentially Expand and Mediate Pathologic Alloreactivity in Acute Graft Versus Host Disease

Blood ◽

10.1182/blood.v118.21.1972.1972 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1972-1972

Author(s):

Gerald P. Morris ◽

Geoffrey L Uy ◽

David L Donermeyer ◽

Paul M Allen ◽

John F. DiPersio

Keyword(s):

T Cells ◽

T Cell ◽

Graft Versus Host Disease ◽

Peripheral Blood ◽

Thymic Selection ◽

Cell Repertoire ◽

Host Disease ◽

Graft Versus Host

Abstract Abstract 1972 The nature of the T cell repertoire mediating pathologic in vivo alloreactivity is an important question for understanding the development of acute graft-versus-host disease (aGvHD) following clinical allogeneic transplantation. We have previously demonstrated that the small proportion of T cells that naturally express 2 T cell receptors (TCR) as a consequence of incomplete TCRa allelic exclusion during thymic development contribute disproportionately to the alloreactive T cell repertoire, both in vitro and in vivo in a mouse model of graft versus host disease (GvHD) (J. Immunol., 182:6639, 2009). Here, we extend these findings to human biology, examining dual TCR T cells from healthy volunteer donors (n = 12) and patients who have undergone allogeneic hematopoietic stem cell transplantation (HSCT) (n = 19). Peripheral blood was collected at day 30 post-HSCT or at the time of presentation with symptomatic acute GvHD. Dual TCR T cells were measured in peripheral blood by pair-wise staining with 3 commercially-available and 2 novel TCRa mAbs. Dual TCR T cells were consistently and significantly expanded in patients with symptomatic aGvHD, representing 5.3±3.8 % of peripheral T cells, compared to 1.7±0.8 % of T cells in healthy controls (p < 0.005) (Figure 1). There was no correlation between dual TCR T cell frequency and GvHD severity. Furthermore, sequential analysis of peripheral blood in 2 patients demonstrated expansion of dual TCR T cells concurrent with the development of aGvHD (Figure 2). Dual TCR T cells from patients with symptomatic aGvHD demonstrated increased expression of CD69 as compared to T cells expressing a single TCR, indicative of preferential activation of dual TCR T cells during aGvHD. Similarly, dual TCR T cells isolated from patients with symptomatic aGvHD demonstrate increased production of IFN-g ex vivo, indicative of the ability to mediate pathogenic alloreactive responses. Dual TCR T cell clones isolated from healthy donors and patients post-HSCT by single cell FACS sorting demonstrate alloreactive responses against a range of allogeneic cell lines in vitro. We propose that the increased alloreactivity of dual TCR T cells results from the less stringent thymic selection for secondary TCR, and thus provides a link between thymic selection, the TCR repertoire, and alloreactivity. These findings may lead to simple ways of phenotypically identifying specific T cells predisposed to inducing aGvHD for subsequent examination of T cell repertoires and functional studies. Furthermore, these data suggest that dual TCR T cells represent a potential predictive biomarker for aGvHD and a potential target for selective T cell depletion in HSCT. Disclosures: No relevant conflicts of interest to declare.

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Chimerism, Lymphocyte Recovery, and the Absence of Graft-Versus-Host Disease in Recipients of Mismatched Unrelated Combined Kidney and HSC Transplants for Tolerance Induction

Blood ◽

10.1182/blood.v118.21.1969.1969 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1969-1969

Author(s):

Joseph Leventhal ◽

Paul A Cardenas ◽

Mary J Elliott ◽

Suzanne T Ildstad

Keyword(s):

T Cells ◽

Stem Cell ◽

Graft Versus Host Disease ◽

Peripheral Blood ◽

Living Donor ◽

Renal Transplant Recipients ◽

Host Disease ◽

Graft Versus Host ◽

Post Transplant

Abstract Abstract 1969 Background: It has been known for over 50 years that hematopoietic stem cell (HSC) chimerism induces tolerance to transplanted tissues and cells. However, the widespread application of this approach has been constrained by graft-versus-host disease (GVHD), the need for close genetic matching between donor and recipient, and the toxicity of conditioning the recipient to establish chimerism. We have demonstrated that full donor chimerism can be established with minimal toxicity in highly-mismatched unrelated and related kidney allograft recipients through nonmyeloablative conditioning followed by infusion of a bioengineered CD8+/TCR− facilitating cell stem cell graft (FCRx), to avoid the risk of GVHD while achieving chimerism. Methods: Twelve HLA-mismatched living donor renal transplant recipients have been entered into a phase 2 trial (IDE 13947) involving low-intensity conditioning (fludarabine, cyclophosphamide, 200 cGy TBI days −4 to −1). Patients received a living donor kidney transplant on day 0, followed by infusion of G-CSF cryopreserved FCRx on day +1. All subjects were discharged by post-operative day 3 and managed as outpatients. We herein present data regarding the immunologic recovery observed in our first 8 evaluable patients with > 6 months follow up. Results: All patients experienced an expected nadir period affecting leukocytes (ANC < 500, range 2–14 days) and platelets (< 50K, range 0–20 days). All patients demonstrated peripheral blood macrochimerism at 1 month post-transplant, ranging from 6% to 100%. Chimerism was gradually lost in two patients at 3 and 6 months post-transplant. Patients demonstrated in vitro evidence of donor-specific hyporesponsiveness (DSH) by MLR +/− CML as early as 3 months post-transplant; of interest, DSH also was observed and persisted in the two patients who lost peripheral blood chimerism. Patients at > 1 yr post-transplant are immunocompetent to respond to mitogen (PHA), MHC-disparate third-party alloantigen, and tetanus in in vitro proliferative assays. Immunologic reconstitution in kidney + FCRx recipients was characterized by a blunted return in CD4+ T cells, with inversion of the CD4/CD8 ratio. A preferential recovery of memory (CD4+/CD45RO+/CD62L+/−) vs. naïve (CD4+/CD45RA+/CD62L+) T cells was observed. Although total number of CD4+/CD25+/CD127lo/FoxP3+ Treg was reduced initially, an increase in the CD4+ Treg /CD4+Teff (CD4+/CD45RA+/CD62L−) ratio was seen in patients exhibiting durable chimerism. In addition, an expansion of central memory CD8+ T cells was observed in durably chimeric recipients. No patient developed donor-specific antibodies as assessed by flow cytometric analysis. The absence of GVHD correlated with in vitro hyporesponsiveness of the fully chimeric recipients against archived pre-treatment recipient APC. Conclusions: Combined kidney + FCRx recipients demonstrate characteristic immunophenotypic and functional changes associated with reconstitution following transplantation; additional studies are required to determine whether these changes are mechanistically related to the persistence of chimerism and/or prevention of GVHD. Disclosures: Ildstad: Regenerex LLC: Equity Ownership.

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Human Mesenchymal Stem Cells Attenuate Graft-Versus-Host Disease and Maintain Graft-Versus-Leukemia in Murine Allogeneic Bone Marrow Transplantation

Blood ◽

10.1182/blood.v118.21.1907.1907 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1907-1907 ◽

Cited By ~ 1

Author(s):

Jeffery J Auletta ◽

Saada Eid ◽

Matthew Keller ◽

Leland Metheny ◽

Rocio Guardia-Wolff ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Transplantation ◽

Graft Versus Host Disease ◽

Marrow Transplantation ◽

Allogeneic Bone Marrow Transplantation ◽

Allogeneic Bone ◽

Graft Versus Host ◽

Graft Versus Leukemia

Abstract Abstract 1907 Defining in vivo effects and biodistribution of human bone marrow-derived mesenchymal stem cell (hMSCs) following allogeneic bone marrow transplantation (alloBMT) could impact the clinical utility of MSC therapy for the prevention and treatment of graft-versus-host disease (GvHD). Using an established model of murine alloBMT, we defined hMSC effects on GvHD and graft-versus-leukemia (GvL) activity. We first studied whether hMSC could modulate in vitro murine T-cell (TC) alloreactivity in mixed leukocyte cultures (MLCs). Specifically, hMSCs added to MLCs significantly reduced TC proliferation in a concentration-dependent manner distinct from human fibroblasts. In contrast to MLC cultures alone, MLCs containing hMSCs had significant reduction in TNFα, IFNγ, and IL-10 levels and higher levels of PGE2 and TGFβ1. Modulation in the inflammatory milieu was associated with changes in TC phenotypes, including more naïve and less activated TC surface marker expression (CD62L+CD69−) and the induction of CD4+CD25+FoxP3+ T-regulatory cells. To determine whether hMSCs could modulate in vivo mTC alloreactivity, irradiated recipient B6D2F1 (H-2bxd) mice were transplanted with allogeneic C57BL/6 (H-2b) BM and purified splenic TCs (B6→B6D2F1) and then were tail-vein injected with hMSC infusions (1 million per injection) on days one and four post-transplant. Syngeneic transplant recipients (B6D2F1→B6D2F1) were used as controls. hMSC-treated alloBMT mice had significantly prolonged survival and improved clinical GvHD scores, reduced splenic TC expansion and TNFα and IFNγ-producing TCs, and lower circulating TNFα and IFNγ levels versus untreated alloBMT mice. Bioluminescence imaging showed redistribution of labeled hMSCs from the lungs to abdominal organs within 72 hours following infusion. Importantly, GvHD target tissues (small and large bowel and liver) harvested from hMSC-treated alloBMT mice had significantly lower GvHD pathology scores than untreated alloBMT mice. We next determined the effects of hMSCs on GvL activity using the murine mastocytoma cell line, P815 (H-2d). TCs co-cultured with hMSCs maintained potent in vitro cytotoxic T-lymphocyte (CTL) activity comparable to untreated control CTLs. After challenge with P815 tumor cells, hMSCs-treated alloBMT mice had less severe GvHD, eradication of tumor burden, and improved leukemia-free survival compared to alloBMT control mice. Lastly, indomethacin (IM) added to MLC-hMSC co-cultures significantly reversed attenuation in both murine TC alloreactivity and surface activation expression. In addition, IM administered to hMSC-treated alloBMT mice reversed hMSC-associated survival advantage, suggesting that PGE2 in part mediates hMSC immunomodulatory effects. Together, our results show that hMSC infusions effectively attenuate GvHD and maintain GvL potency in alloBMT mice and reveal potential biomarkers and mechanisms of action underlying hMSC effects. Disclosures: Solchaga: Bimemetic Therapeutics: Employment. Cooke:Amgen: Provides experimental drug and central pharmacy support for 2 trials for which I am Co-PI.

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Donor Requirements For CD4+CD25+FoxP3+ Regulatory T Cells Capable Of Suppressing CD4+ and CD8+ Conventional T Cell Proliferation and Graft Versus Host Disease

Blood ◽

10.1182/blood.v122.21.4484.4484 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4484-4484 ◽

Cited By ~ 1

Author(s):

Antonio Pierini ◽

Lucrezia Colonna ◽

Maite Alvarez ◽

Dominik Schneidawind ◽

Byung-Su Kim ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Animal Models ◽

Graft Versus Host Disease ◽

Third Party ◽

T Cell Proliferation ◽

Graft Versus Host

Adoptive transfer of CD4+CD25+FoxP3+ regulatory T cells (Tregs) prevents graft versus host disease (GvHD) in several animal models and following allogeneic hematopoietic cell transplantation (HCT) in clinical trials. In these models donor derived Tregs have been mainly used as they share the same major histocompatibility complex (MHC) with conventional CD4+ and CD8+ T cells (Tcons) that are primarily responsible for GvHD onset and persistence. Third-party derived Tregs are a promising alternative tool for cellular therapy as they can be prepared in advance, screened for pathogens and activity and banked. In this study we explored MHC disparities between Tregs and Tcons in HCT to evaluate the impact of these different cell populations in GvHD prevention and survival after transplant. Methods and Results We evaluated the ability of highly purified Treg to suppress proliferation of C57BL/6 (H-2b) Tcons following exposure to irradiated splenocytes from BALB/C (H-2d) mice in vitro in a mixed lymphocyte reaction (MLR). Either donor derived C57BL/6 (H-2b) or third party FVB (H-2q) Tregs suppressed Tcon proliferation at the Treg/Tcon ratios of 1:2 and 1:4. The same Treg population effectively suppressed different MHC derived Tcons where BALB/C (H-2d) or FVB (H-2q, third-party) Tcons were incubated with irradiated splenocytes from C57BL/6 (H-2b) mice and were effectively suppressed with BALB/C (H-2d) Tregs. In the MLR, third-party Tregs present the same activation molecule expression patterns as MHC matched Tregs: CTLA4 and LAG3 expression is enhanced after stimulation with interleukin-2 (IL-2) and anti-CD3/CD28 beads, while MHC class II molecule expression is increased after 3-4 days of culture with Tcons and irradiated splenocytes. Furthermore third-party and MHC matched Tregs express the same levels of interleukin-10 (IL-10). We translated these results to in vivo studies in animal models. In these studies T cell depleted bone marrow (TCD BM) from C57BL/6 (H-2b) mice was injected into lethally irradiated (total body irradiation, 8 Gy) BALB/C (H-2d) recipient mice. 2 days later GvHD was induced by injecting luc+ donor derived Tcons (1x106/mouse). Using this model GvHD was evaluated following the adoptive transfer of freshly isolated CD4+CD25+FoxP3+ Tregs derived from BALB/C (H-2d, host type), C57BL/6 (H-2b, donor type), FVB (H-2q, third-party) or BALB/B (H-2b, minor mismatched with the donor, major mismatched with the host) mice at the different Treg/Tcon ratios of 1:1, 1:2 and 1:4. As expected, donor Tregs exerted the strongest dose dependent GvHD protection (p = 0.028), while host Tregs did not improve mouse survival (p = 0.58). Third-party and minor mismatched with the donor Tregs improved mouse survival (third-party and minor mismatched with the donor respectively, p = 0.028 and p = 0.17) but mice had worse GvHD score profiles (both p< 0.001) and could not recover their weight as well as mice treated with donor Tregs (both p< 0.001). In vivoTcon bioluminescent imaging confirmed these results showing a reduced Tcon proliferation in mice treated with donor, third-party and minor mismatched with the donor Tregs, the first exerting the strongest effect (after 6 weeks of observation, p< 0.001). Conclusions Our studies indicate that MHC disparities between Tregs and Tcons do not represent an insurmountable barrier for Treg function. In vitro and in vivo data strongly suggest that Tregs can suppress Tcon proliferation without requiring MHC matching. In vivo GvHD prevention efficiency was affected by MHC disparities with donor derived Treg being the most effective, however, third party Treg also resulted in GvHD attenuation. These studies indicate that both donor and third party Treg could be effective in clinical application raising the possibility of screening and banking Treg for use. Further, these studies highlight the need for activation of the Treg on host tissues to effectively suppress conventional T cell proliferation and GvHD induction. Disclosures: No relevant conflicts of interest to declare.

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G-CSF as immune regulator in T cells expressing the G-CSF receptor: implications for transplantation and autoimmune diseases

Blood ◽

10.1182/blood-2002-04-1200 ◽

2003 ◽

Vol 102 (2) ◽

pp. 734-739 ◽

Cited By ~ 130

Author(s):

Anke Franzke ◽

Wenji Piao ◽

Jörg Lauber ◽

Patricia Gatzlaff ◽

Christian Könecke ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

Autoimmune Diseases ◽

Graft Versus Host Disease ◽

Bone Marrow Failure ◽

Host Disease ◽

Graft Versus Host ◽

Acute Graft

Abstract Results from experimental models, in vitro studies, and clinical data indicate that granulocyte colony-stimulating factor (G-CSF) stimulation alters T-cell function and induces Th2 immune responses. The immune modulatory effect of G-CSF on T cells results in an unexpected low incidence of acute graft-versus-host disease in peripheral stem cell transplantation. However, the underlying mechanism for the reduced reactivity and/or alloreactivity of T cells upon G-CSF treatment is still unknown. In contrast to the general belief that G-CSF acts exclusively on T cells via monocytes and dendritic cells, our results clearly show the expression of the G-CSF receptor in class I– and II– restricted T cells at the single-cell level both in vivo and in vitro. Kinetic studies demonstrate the induction and functional activity of the G-CSF receptor in T cells upon G-CSF exposure. Expression profiling of T cells from G-CSF–treated stem cell donors allowed identification of several immune modulatory genes, which are regulated upon G-CSF administration in vivo (eg, LFA1-α, ISGF3-γ) and that are likely responsible for the reduced reactivity and/or alloreactivity. Most importantly, the induction of GATA-3, the master transcription factor for a Th2 immune response, could be demonstrated in T cells upon G-CSF treatment in vivo accompanied by an increase of spontaneous interleukin-4 secretion. Hence, G-CSF is a strong immune regulator of T cells and a promising therapeutic tool in acute graft-versus-host disease as well as in conditions associated with Th1/Th2 imbalance, such as bone marrow failure syndromes and autoimmune diseases.

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Damage to Thymic Stroma Results in Increased Production of Pathogenic Dual Receptor T Cells Associated with Chronic Graft Versus Host Disease

Blood ◽

10.1182/blood.v128.22.1156.1156 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1156-1156

Author(s):

Amritha Balakrishnan ◽

Burhan Jama ◽

Nicholas Joseph Gloude ◽

Eric Jon Anderson ◽

Edward D. Ball ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Single Cell ◽

Graft Versus Host Disease ◽

Graft Versus Host ◽

Post Transplant ◽

Thymic Stroma

Abstract Evidence from clinical investigations and animal models indicate that chronic graft versus host disease (cGVHD) results from defective thymic generation of functional and self-tolerant T cell populations following hematopoietic stem cell transplantation (HSCT). We have previously demonstrated that the rare subset of T cells that naturally express 2 T cell receptors (TCRs) on the cell surface as a result of incomplete allelic exclusion are predisposed to respond to auto- and alloantigens. Dual TCR T cells disproportionately participate in pathologic alloreactivity in HSCT patients and mouse models of acute GVHD. These findings, combined with observations demonstrating that dual TCR T cells represent a physiologic reservoir of unique TCRs that evade negative selection, prompted us to examine the role of thymic selection and dual TCR T cells in cGVHD. To study the role of post-transplant thymopoiesis in generation of potentially pathogenic dual TCR T cells, we used a mouse model of syngeneic bone marrow transplantation into lethally-irradiated recipients. Radiation-induced damage to the thymic stroma was characterized by disruption of thymic architecture and loss of cortical and medullary thymic epithelial cells (TECs). This damage resulted in significantly increased generation of dual TCR T cells following transplantation of congenically-marked syngeneic T cell-depleted bone marrow. Two-fold increased production of dual TCR T cells persisted for at least 20 weeks after transplantation. These data demonstrate the hazard for production of T cells predisposed to pathogenic reactivity in the post-transplant environment, and suggest that dual TCR T cells could be a source of T cells causing cGVHD. To examine involvement of dual TCR T cells in cGVHD, we analyzed peripheral blood samples from patients after allogeneic HSCT (> 12 months post-transplant) using our previously utilized pair-wise TCRVa labeling flow cytometry approach. Flow cytometry analysis revealed that dual TCR T cells were present at increased frequencies in patients with cGVHD (n = 10, 8.3% + 1.1%, P = 0.028) compared to patients without cGVHD (n = 3, 2.5 + 1.1%) or healthy age-matched controls (n = 5, 1.9 + 0.4%). Dual TCR T cells from patients with cGVHD had an activated CD69+ phenotype as compared to T cells expressing only a single TCR from the same patient. Single-cell TCRa/TCRb sequencing confirmed the increased frequencies of dual TCR T cells specific to activated T cells in patients with cGVHD. Repertoire analysis of TCRs sequenced from single cells indicated that the increase in dual TCR T cells was polyclonal. The single-cell sequencing approach enabled multiplexed examination of T cell lineage-associated transcription factors and cytokines. Single-cell transcriptional profiling demonstrated that dual TCR T cells demonstrated predominantly pro-inflammatory and cytotoxic phenotypes with expression of Tbet and perforin. This is in contrast to T cells expressing only a single TCR from the same patient, or dual TCR T cells from healthy control patients, which had a quiescent phenotype. These data indicate a role for dual TCR T cells in mediating cGVHD. Together, these results suggest that dual TCR T cells may be an important link between post-transplant T cell development and cGVHD. Disclosures No relevant conflicts of interest to declare.

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