scholarly journals Quantity and Quality Reconstitution of NKG2A+ NK Cells Are Associated with Graft-Versus-Host Disease after Allogeneic Hematopoietic Cell Transplantation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4582-4582
Author(s):  
Lijuan Hu ◽  
Xiang-Yu Zhao ◽  
Xingxing Yu ◽  
Meng Lv ◽  
Ting-Ting Han ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Graft Versus Host Disease ◽  
Acute Gvhd ◽  
Nk Cell ◽  
Graft Versus Host ◽  
Cell Counts ◽  
Alloreactive T Cells

Abstract Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment strategy for hematological malignancies. However, graft-versus-host disease (GVHD) is a common complication after allo-HSCT resulting from the activation, amplification and secretion of numerous inflammatory factors related to donor alloreactive T cells that damage host tissues and organs, mainly in the gastrointestinal tract, liver and skin. Notably, natural killer (NK) cells represent the first donor-derived lymphocyte population to recover after allo-HSCT and generally are observed within the first month after allo-HSCT. NK cells express a series of immune receptors that identify relevant ligands on target cells and maintain the immune balance between NK cell activation and tolerance. Previous studies have shown that the number of NKG2A+ cells is decreased in patients with chronic GHVD after HSCT. However, the relationship between NKG2A+ NK cells and aGVHD has not been characterized. In addition, the role of NKG2A+ cells in aGVHD disease progression and the mechanism underlying NKG2A+ cell immunoregulation have not been clearly explained. Objective: In this study, we used peripheral blood from GVHD, non-GVHD paired specimens and healthy donors to address the underlying mechanism by which NKG2A+ NK cells regulate T cells after HSCT. Methods: We detected the specimens using flow cytometry from two independent cohorts, which were prospective cohort and paired cohort. Futher, we performed experiments in vitro to investigate the potential role of NKG2A+ NK cells on T cells. Results: Here, we found that, compared with non-GVHD subset, NKG2A+ NK cells percentage and absolute cell counts were significantly reduced in GVHD patients after HSCT. Moreover, the reduction of NKG2A+ NK cells in GVHD patients was ascribed to its increased apoptosis and decreased proliferation capacity, while retaining a strong graft-versus-leukemia (GVL) effect. In vitro assay showed that when co-cultured T cells with NKG2A+ NK cells, the T cells secreted IFN-r level was significantly reduced, while the IL-4 level was increased. Moreover, CD25 expression level was decreased, while the CD4+CD25+FOXP3+ cells number was increased. In addition, the NKG2A+ NK cells induced T cell apoptosis and decreased T cell proliferation during the coculture process. Significantly, NKG2A+ mainly regulated activated but not resting T cells. In vivo assay showed that serological IL-10 level in GVHD subset was evidently lower than those of non-GVHD subgroup, the IL-1β, IFN-r and TNF-a level was however higher in the GVHD subgroup. Furthermore, the percentage of NKG2A+ NK cells from GVHD patients was markedly increased by the presence of exogenous IL-10, but not by other cytokines. However, this phenotype was not observed at non-GVHD patients. Together, the GVHD might be ascribed to lower IL-10 induced NKG2A+ NK cells reduction, which further overactivate T cells after HSCT. Discussion and Conclusion: Overall, we herein observed reduced proportions and absolute cell counts of NK cells and NKG2A+ subsets in patients with acute GVHD after allo-HSCT. The causative association between NK cell numbers, NKG2A+ subsets and GVHD remains debatable. Based on our results, speculating that reduced proportions of NKG2A+ subsets in patients after allo-HSCT are associated with acute GVHD due to their interplay with the patient's donor-derived alloreactive T cells is tempting. Disclosures No relevant conflicts of interest to declare.

scholarly journals Microrna-191 Regulates T-Cell Clonal Expansion during Graft-Versus-Host Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4433-4433
Author(s):  
Chuanfeng Xiong ◽  
Wei Huang ◽  
Xiaoli Nie ◽  
Ying Huang ◽  
Yiqun Jiao ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Graft Versus Host Disease ◽  
Clonal Expansion ◽  
Host Disease ◽  
Graft Versus Host ◽  
Alloreactive T Cells

Allogeneic hematopoietic cell transplantation is a potentially curative treatment choice for a wide variety of hematological malignancies. However, graft-versus-host disease (GVHD), which is mediated by donor alloreactive T cells, limits the success of this procedure. Previous studies have demonstrated that several microRNAs (miRs) modulate graft-versus-host disease. miR-191 was previously reported to be able to support T cell survival after TCR stimulation. We hypothesize that miR191 regulates T cell response during GVHD. To test this hypothesis, we first studied miR-191 expression in alloreactive T cells. The result demonstrated that miR-191 was up-regulated in donor T cells isolated from murine GVHD recipients, suggesting that miR-191 may play a role in GVHD induction. We further studied the role of miR-191in GVHD using miR-191 deficient T cells (KO). Lethally irradiated (8.5 Gy) BALB/c mice were injected intravenously with 1×107 T cell-depleted bone marrow (TCDBM) cells along with 1×106 purified T cells from wild-type (WT) or KO mice, which are in C57BL/6 background. Interestingly, all recipients in the WT group died within 35 days after transplantation, while only one out of ten animals died in the KO group during an observation period of 56 days. Body weights and clinical scores were also improved in KO T cell recipients when compared with the WT controls. Similar results were also observed in a second GVHD model (C57BL/6→C3H/HeJ). To understand the mechanism by which miR-191 KO T cells have decreased ability to mediate GVHD, we first measured the ability of KO T cells to respond to alloantigens in vitro in a mixed lymphocytes reaction assay. Dramatically decreased alloresponse was observed with KO T cells as compared with WT T cells. Similarly, decreased clonal expansion was observed in KO T cells in vivo upon challenge with alloantigens as measured by bioluminescent imaging (Figure 1A). These results were further supported by data from a co-transfer experiment, in which equal numbers of WT and KO T cells were transplanted into the same GVHD recipient. At day7 after transplantation, KO T cells showed significantly reduced expansion in the spleen and liver compared with WT T cells. Reduced alloresponses mediated by KO T cells may not due to decreased proliferative capability directly as an in vivo carboxyfluorescein succinimidyl ester (CFSE) assay showed a comparable cell division between WT and KO T cells upon challenge with alloantigens. Rather, increased cell death is responsible for decreased alloresponse observed in KO T cells because dramatically increased number of dead cells was observed in KO group compared with WT group upon response to alloantigens in vitro and vivo. To determine the genes that are regulated by miR-191, we did a screening based on the prediction. Humans and mice share more than 100 predicted targets for miR-191. We chose top 20 of these targets for RT-qPCR screening. The result demonstrated that Taf5 was a target gene of miR-191. Expression of TAF5 protein was down-regulated in activated KO T cells when compared with the WT T cells. Finally, we investigated whether miR-191 KO T cells preserve graft-versus-leukemia effects. 1×106 T cells from WT or KO mice were transplanted into lethally irradiated BALB/c mice along with 1×107 TCDBM cells and 1×105 host-type BCL-1 cells. While all recipients that received only TCDBM and tumor cells developed lethal leukemia/lymphoma, none of WT and KO T cells recipients developed tumor. In conclusion, our findings reveal a critical role of miR-191 during GVHD process and demonstrate that miR-191 is a novel therapeutic target for GVHD. Figure 1 Disclosures No relevant conflicts of interest to declare.

scholarly journals HLA-E Up-Regulation Induced by HIV Infection May Directly Contribute to CD94-Mediated Impairment of NK Cells

2005 ◽  
Vol 18 (2) ◽  
pp. 269-276 ◽  
Author(s):  
F. Martini ◽  
C. Agrati ◽  
G. D'Offizi ◽  
F. Poccia
Keyword(s):  
T Cells ◽  
Hiv Infection ◽  
Nk Cells ◽  
Cd4 T Cells ◽  
Nk Cell ◽  
Treatment Interruption ◽  
Cell Number ◽  
Hiv Replication

Alterations in NK cell numbers and function have been repeatedly shown during HIV infection. In this study, NK cell number and MHC class I expression on CD4+ T cells were studied in HIV patients at different stages of disease progression. An increased expression of HLA-E was seen on CD4+ T cells. In parallel, a reduced number of CD94+ NK cells was observed in advanced disease stages. Moreover, a decline in CD94 expression on NK cells was observed at the HIV replication peak in patients undergoing antiretroviral treatment interruption, suggesting a role of viral replication on NK cells alterations. In vitro HIV infection induced a rapid down-regulation of HLA-A,B,C expression, paralleled by an increased expression of HLA-E surface molecules, the formal ligands of CD94 NK receptors. HIV-infected HLA-E expressing cells were able to inhibit NK cell cytotoxicity through HLA-E expression, since cytotoxicity was restored by antibody masking experiments. These data indicate that the CD94/HLA-E interaction may contribute to NK cell dysfunction in HIV infection, suggesting a role of HIV replication in this process.

A combination of anti-CD3 and anti-CD7 ricin A-immunotoxins for the in vivo treatment of acute graft versus host disease

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3693-3701 ◽  
Author(s):  
Ypke V. J. M. van Oosterhout ◽  
Liesbeth van Emst ◽  
Anton V. M. B. Schattenberg ◽  
Wil J. M. Tax ◽  
Dirk J. Ruiter ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Graft Versus Host Disease ◽  
T Cell Response ◽  
Cytotoxic T Cell ◽  
Host Disease ◽  
Graft Versus Host ◽  
Ricin A

Abstract This study evaluated the anti-graft versus host disease (GVHD) potential of a combination of immunotoxins (IT), consisting of a murine CD3 (SPV-T3a) and CD7 (WT1) monoclonal antibody both conjugated to deglycosylated ricin A. In vitro efficacy data demonstrated that these IT act synergistically, resulting in an approximately 99% elimination of activated T cells at 10−8 mol/L (about 1.8 μg/mL). Because most natural killer (NK) cells are CD7+, NK activity was inhibited as well. Apart from the killing mediated by ricin A, binding of SPV-T3a by itself impaired in vitro cytotoxic T-cell cytotoxicity. Flow cytometric analysis revealed that this was due to both modulation of the CD3/T-cell receptor complex and activation-induced cell death. These results warranted evaluation of the IT combination in patients with refractory acute GVHD in an ongoing pilot study. So far, 4 patients have been treated with 3 to 4 infusions of 2 or 4 mg/m2 IT combination, administered intravenously at 48-hour intervals. The T1/2 was 6.7 hours, and peak serum levels ranged from 258 to 3210 ng/mL. Drug-associated side effects were restricted to limited edema, fever, and a modest rise of creatine kinase levels. One patient developed low-titer antibodies against ricin A. Infusions were associated with an immediate drop of circulating T cells, followed by a more gradual but continuing elimination of T/NK cells. One patient mounted an extensive CD8 T-cell response directly after treatment, not accompanied with aggravating GVHD. Two patients showed nearly complete remission of GVHD, despite unresponsiveness to the extensive pretreatment. These findings justify further investigation of the IT combination for treatment of diseases mediated by T cells.

Toll-Like Receptors 2 and 4 Are Involved in the Induction of Graft-Versus-Host-Disease in Mice.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5175-5175
Author(s):  
Axel Nogai ◽  
Markus M. Heimesaat ◽  
Marc Thiele ◽  
Stefan Bereswill ◽  
Eckhard Thiel ◽  
...  
Keyword(s):  
T Cells ◽  
Graft Versus Host Disease ◽  
Lipid A ◽  
Host Disease ◽  
Graft Versus Host ◽  
Vitro Data ◽  
In Vitro Data

Abstract BACKGROUND: Intestinal Graft-versus-Host disease is a frequent and often lethal complication after allogenic stem cell transplantation. Since NOD2 polymorphisms have been recognized as potential triggers of severe intestinal GvHD in humans, we have developed murine transplantation models to investigate the role of different pattern recognition receptors (PRR) in GvHD and GvL. Here we report our results on the role of TLR2 and TLR4 for the induction of GvHD. METHODS: Severity of GvHD in wildtype (wt) C57B/10 (H-2Db), TLR2−/−, TLR4−/−, and combined TLR2−/−TLR4−/− C57B/10 mice was investigated. Mice received treosulfan 2000 mg/kg from day -3 to -1 and cyclophosphamide 200 mg/kg day -1 prior to injection of 10×10^6 H-2Dd BM cells and 5×10^6 splenocytes (SC). Survival and GvHD score were assessed daily. Engraftment was determined every 2 weeks in pB and at the end of the experiments in bone marrow by flow cytometry. T cell alloreactivity in GvH direction was assessed by MLR using splenocytes as stimulators from PRR-deficient mice or wt as control and CFSE-staining as read-out. The relevance of PRR ligands for the enhancement of GvH alloreactivity was determined by addition of lipid A, lipopetides, or CpG. RESULTS: in vivo data: The transfer of 10×10^6 BMC + 5×10^6 SC induced a severe GvHD in all wt recipients, leading to death of 90% of the animals within 20 days. Recipient mice lacking either TLR2 or TLR4 showed only a slightly and not significantly decreased GvHD lethality. In recipients lacking both PPRs, i.e. TLR2 and TLR4, GvHD was generally milder and the majority (60%) of the animals survived until day 20 (p<0.05). However, the long term survival was not significantly improved. Differences in clinical severity of GvHD were confirmed histologically. In vitro data: Stimulation with cells from TLR2−/− and TLR4−/− mice resulted in a decreased alloreactivity in MLR. A median of 2% of Balb/c CD4+ T cells proliferated in response to C57B/10 stimulators. The addition of the TLR2 and TLR4 ligands lipopeptide, Lipid A and CpG significantly (p<0.05) increased the proliferation of CD4+ T cells in a specific manner more than twofold. CONCLUSION: Our in vivo and in vitro data consistantly show that bacterial components are involved in triggering GvH alloreactivity via different types of PPRs. Binding of bacterial substances to TLR2 and TLR4 leads to activation of the immune system and subsequent induction of GvHD. Our data provide an experimental basis for the development of strategies for modulation of the intestinal gut flora by selective gut decontamination and/or probiotic regimens to prevent GvHD in humans.

Efficient Treatment of Murine Acute Graft-Versus-Host Disease with In Vitro Expanded CD4+CD25+ Regulatory T Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2987-2987
Author(s):  
Tina J Boeld ◽  
Kristina Doser ◽  
Corinna Lang-Schwarz ◽  
Elisabeth Huber ◽  
Reinhard Andreesen ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Regulatory T Cells ◽  
Graft Versus Host Disease ◽  
Acute Gvhd ◽  
Treg Cells ◽  
Host Disease ◽  
Graft Versus Host ◽  
Acute Graft

Abstract Abstract 2987 Acute graft-versus-host disease (GVHD) is a frequent complication after allogeneic bone marrow transplantation (BMT). We previously showed that the adoptive transfer of donor-type CD4+CD25+ regulatory T cells (Treg) at the time of BMT prevents acute GVHD in murine models. However, the therapeutic potential of donor-derived Treg cells for the treatment of established acute GVHD has not yet been examined in detail. In analogy to potential clinical applications we now tested the capacity of in vitro expanded Treg cells to ameliorate acute GVHD after haploidentical BMT (BALB/c→CB6F1). CD4+CD25highCD62L+ Treg cells were purified by FACS and stimulated polyclonally using anti-CD3/CD28-coated beads. Cells expanded on average 130±19-fold (n=7) within 2 wks and maintained high levels of FoxP3 expression (96, 8±0, 8% FoxP3+ cells; n=7) as well as potent immunosuppressive activity in vitro. For the induction of acute GVHD CB6F1 recipients were lethally irradiated and transplanted with 2.5×106 BM cells in combination with 5×106 splenocytes. All animals developed severe GVHD by d11, as revealed by an increase of the GVHD severity score (2.3±0.4 in GVHD animals vs 0±0 in BM controls, p<0.001, n=1–11) and by histological analyses of the gut (score: 7.8±0.4 for the GVHD group vs 0.2±0.2 for BM controls, p =0.046, n=3). When animals with acute GVHD were treated with 5×106 expanded CD4+CD25highCD62L+ Treg cells on d11 after BMT, they initially developed progressive GVHD comparable to non-treated GVHD animals, as indicated by weight loss and an increase of the GVHD score. However from d44 post BMT onwards, Treg-treated GVHD animals regained body weight (d44: 75±3% vs 67±2% of initial weight; p <0.05; n=9–10) and their clinical GVHD score (d44: 6±0 vs 4.3±0.4; p <0.05; n=9–10) decreased. While all non-treated GVHD animals succumbed to disease by d67 after transplantation, 50% of Treg-treated GVHD animals survived for at least 100d (p =0, 002; n=16–21). As immune reconstitution and in particular reconstitution of the lymphocyte compartment is impaired in animals with GVHD, we analyzed the effect of Treg therapy on the reconstitution of the lymphoid and myeloid compartment. At d21 after BMT spleen and BM of non-treated as well as Treg-treated GVHD animals were completely lymphopenic as compared to control mice and both organs contained exceptionally high numbers of granulocytes. Unlike non-treated GVHD animals, however, Treg-treated recipients by d60 showed a recovery of the lymphocyte compartment in spleen (10±2.6×106 T cells and 23.5±12.5×106 B cells in Treg-treated vs 3.0±0.6×106 T cells and 1.5±0.4×106 B cells in non-treated GVHD animals vs 26.25±2.6×106 T cells and 63.9±9.1×106 B cells in BM controls) and BM (0.7±0.1×106 T cells and 8.6±4×106 B cells in Treg-treated vs 0.3±0.01×106 T cells and 0.7±0.4 ×106 B cells in non-treated GVHD animals vs 0.4±0.03×106 T cells and 11.2±0.6×106 B cells in BM controls), while the number of granulocytes decreased constantly. Successful treatment with Treg cells was finally accompanied by a reconstitution of the lymphatic system comparable to control mice. Furthermore, successfully treated mice showed only mild histological signs of gut GVHD at d100 that was significantly lower then those in non-treated GVHD animals with end-stage disease (score: 4.2±1 vs 9.9±1.5 in treated vs non-treated animals, p =0.006, n=4–6). Taken together, these results indicate that in vitro expanded natural Treg cells may not only be effective for the prevention, but also for the treatment of acute GVHD after allogeneic BMT. Disclosures: No relevant conflicts of interest to declare.

MiR-146a Regulates the TRAF6/TNF-Axis in Donor T Cells during Graft-Versus-Host Disease

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 843-843
Author(s):  
Natalie Stickel ◽  
Gabriele Prinz ◽  
Dietmar Pfeifer ◽  
Annette Schmitt-Graeff ◽  
Marie Follo ◽  
...  
Keyword(s):  
T Cells ◽  
Graft Versus Host Disease ◽  
Acute Gvhd ◽  
Negative Regulator ◽  
Snp Analysis ◽  
Hematopoietic Stem ◽  
Host Disease ◽  
Graft Versus Host ◽  
Donor T Cells

Abstract Introduction: Acute graft-versus-host disease (GvHD) arises from the attack of recipient tissues by donor allogeneic T cells and represents one of the major limitations of allogeneic hematopoietic cell transplantation (allo-HCT). In spite of many clinical trials, the standard immunosuppressive regimens for prevention of acute GvHD have improved little in the last two decades. Hence, a better understanding of the biology of acute GvHD may improve therapeutic options. MicroRNA-146a (miR-146a) was found to be increased in the sera of patients with GvHD. Therefore, we aimed to decipher the role of miR-146a in allogeneic donor T cells during GvHD by functional studies and in patients undergoing allo-HCT by single nucleotide polymorphism (SNP) analysis. Methods: We used two different murine major MHC mismatch models for acute GvHD. Recipient mice were conditioned with irradiation before transplantation of bone marrow and either wildtype or miR-146a deficient T cells from allogeneic donor mice. Furthermore, genomic DNA from 289 patients that underwent allo-HCT and their respective hematopoietic stem cell donors was isolated in order to determine their miR-146a rs2910164genotype. Results: We observed miR-146a upregulation in T cells of mice developing acute GvHD compared to untreated mice in a major MHC and a minor histocompatibility antigen mismatch model. Transfer of miR-146a deficient T cells caused increased GvHD severity, elevated TNF serum levels and reduced survival. Conversely, the phytochemical induction of miR-146a or its overexpression in donor T cells using a specific miR-146a mimic reduced GvHD severity. TNF receptor-associated factor 6 (TRAF6), a verified target of miR-146a, was upregulated in miR-146a-/- T cells following alloantigen stimulation. Higher TRAF6 levels translated into increased NF-κB activity and TNF production in miR-146a-/- T cells, while other pro-inflammatory cytokine levels were unaffected. The detrimental effect of miR-146a deficiency in T cells could be antagonized by TNF blockade in vivo. Moreover, in contrast to WT T cells, over expression of miR-146a in Tnf deficient T cells had no effect on their alloreactivity. In the human system, the minor genotype of the SNP rs2910164, which causes reduced miR-146a expression, was more frequent in patients developing acute GvHD grade III/IV compared to all other allo-HCT recipients (n=289). Conclusions: Taken together we show that miR-146a functions as a negative regulator of the TRAF6/TNF-axis in allogeneic donor T cells during GvHD, leading to reduced TNF transcription. Given our observation on the predictive role of the SNP leading to decreased miR-146a expression in acute GvHD in patients and the possibility to exogenously enhance miR-146a expression, we provide a novel and targeted molecular approach to mitigate GvHD. Disclosures No relevant conflicts of interest to declare.

Immunomodulatory Effects of the Triterpenoid CDDO after Allogeneic Bone Marrow Transplantation in Mice: Reduction of Acute Graft-Versus-Host Disease Lethality.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1316-1316
Author(s):  
William J. Murphy ◽  
Olga Frolova ◽  
Marina Konopleva ◽  
Michael Andreeff ◽  
Weihong Ma ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Graft Versus Host Disease ◽  
Acute Gvhd ◽  
Allogeneic Bone ◽  
Host Disease ◽  
Graft Versus Host ◽  
Cancer Relapse ◽  
Allogeneic Bmt

Abstract The use of hematopoietic stem cell transplantation (HSCT) in cancer treatment is seriously hampered by the occurrence of graft-versus-host disease (GVHD) and cancer relapse. During acute GVHD, inflammatory cytokines play a pivotal role in the amplification of GVHD. Therefore, assessment of agents with known anti-neoplastic activity that also reduce cytokine production may be useful in both the prevention of GVHD and cancer relapse. The synthetic triterpenoid, CDDO (2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid) is multifunctional molecule which has shown potent anti-cancer activities both in vitro and in vivo through the induction of apoptosis. We first examined the effects of CDDO on both human and murine T cell mitogen responses in vitro. CDDO significantly inhibited mitogen responsiveness of both human and murine T cells in vitro with evidence of cell cycle arrest of the human T cells. We then proceeded to examine the effects of CDDO on acute GVHD induction and progression. In these studies, lethally irradiated C57BL/6 mice received 10 million bone marrow cells (BMC) and 40 million spleen cells from fully MHC-mismatched BALB/c donors. All of the control mice succumbed rapidly due to acute GVHD. In contrast, the mice that received CDDO (120 ug/BID) given from days 0-3 following BMT exhibited significant improvement in survival (P &lt; 0.001). Body weights from the treated mice also were significantly increased compared to untreated controls. We found that the timing of CDDO administration was a critical factor for protection from GVHD as protection only occurred when CDDO was administered early after BMT. Importantly, donor myeloid reconstitution was not adversely affected by CDDO treatment as determined by peripheral blood cell count and donor chimerism assessment on day +14 post-transplant. No adverse toxicities or effects on reconstitution were observed in the mice receiving BMC alone with CDDO being administered continuously. Given the reported direct anti-tumor effects of CDDO, it will be of particular interest in examining the effects of CDDO and allogeneic BMT in tumor-bearing recipients. Our findings suggest that CDDO can enhance the efficacy of allogeneic BMT by decreasing acute GVHD in mice.

scholarly journals Role of MMP-13 in Graft-Versus-Host Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1928-1928
Author(s):  
Hui-Hui Ma ◽  
Jing Fu ◽  
Suzanne Lentzsch ◽  
Markus Y Mapara
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Dendritic Cells ◽  
Graft Versus Host Disease ◽  
Research Funding ◽  
Host Disease ◽  
Graft Versus Host ◽  
Donor T Cells

Matrix metalloproteinases (MMPs) have been initially recognized for their role in degradation of extracellular matrix (ECM) and collagen remodeling. However, MMPs have been shown to play a crucial role in inflammation, tumor cell invasion, adaptive and innate immunity. Acute and chronic Graft versus Host Disease (GVHD) are characterized by distinctive histopathological features involving tissue infiltration with donor cells, tissue damage and remodeling. We therefore hypothesized that GVHD-associated organ damage may involve MMPs. We have now identified a novel immunomodulatory function for MMP-13 (alternatively called collagenase-3)and have uncovered a previously unknown role of MMP-13 in regulating GVHD.To address the function of MMP-13 in GVHD we first assesed the effect of MMP-13 on alloresponses in vitro. Using fully Major Histocompatibility Complex (MHC)-mismatched standard mixed lymphocyte reaction we demonstated that antigen presentig cells (APC) from B6.MMP-13-/-(H2b) mice led to signifcantly enhanced antigen-driven activation and proliferation of Carboxyfluorescein succinimidyl ester (CFSE)-labeled Balb/c responder splenocytes. Thus, MMP-13 deficiency in either splenocytes or bone marrow-derived dendritic cells used as stimulators resulted in enhanced proliferation, activation and IFN-gproduction in the allo-reactive lymphocyte responders. Similarly, exogenous MMP13 reduced proliferation of responder T cells as determined tested by CFSEdilution (CFSEloof CD4+T cells from 62.3% decreased to 40.6%, CFSEloof CD8+T cells from 74.1% down to 47.9%). We next assessed the impact of MMP-13 in vivousing fully MHC-mismatched rodent acute GVHD models. To study the role of host-derived MMP-13 we induced GVHD in B6.MMP-13-/-or B6.WT recipient mice following lethal TBI (1075 rad) using splenic T cells from Balb/cdonors. We observed signifcantly accelerated GVHD-related mortality (Median Survival Time 7 vs. 47 days post-transplant, p<0.05) in MMP-13-deficient recipients. Most importantly, donor T cells expanded more vigorously in the secondary lymphoid organs (Spleen and mesenteric lymphnoodes) of MMP13-/-compared to wildtype recipient mice (e.g. spleen: absolute donor CD4+Tcells 1.5x104± 7.3 x 103 (WT) vs. 5.83 x104±1.65 x104[MMP-13-/-] and CD8+5.5 x104± 3.8 x104(WT) vs 3.4 x105±1.4 x105[MMP-13-/-], p<0.01). Enhanced donor lymphocyte expansion was further confirmed by bioluminescence imaging. To further delineate the underlying mechanisms, we analyzed the effects of MMP-13deficiency and exogenous MMP-13 on maturation of mouse bone marrow derived-dendritic cells (BMDC) and macrophages in vitro. We noted decreased expression of inhibitory molecules PD-L1 and PD-1H on GM-CSF/LPS cultured BMDC. Similarly, bone marrow-derived MMP-13-/-macrophages also showed reduced PD-L1 and PD-1H expression upon LPS stimulation when compared to their WT counterparts. In summary we posit that recipient myeloid cell-derived MMP-13 mitigates GVHD and limits donor T cell expansion. Further studies are warranted to determine how MMP-13 suppresses expansion of donor T cells and impacts Graft-versus-Leukemia responses. Disclosures Lentzsch: Caelum Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Abbvie: Consultancy; Clinical Care Options: Speakers Bureau; Sanofi: Consultancy, Research Funding; Multiple Myeloma Research Foundation: Honoraria; International Myeloma Foundation: Honoraria; Karyopharm: Research Funding; Columbia University: Patents & Royalties: 11-1F4mAb as anti-amyloid strategy; Proclara: Consultancy; BMS: Consultancy.

Activation of the Adenosine A2A Receptor Promotes the Development of Donor-Derived T Regulatory Cells in a Graft-Versus Host Disease Mouse Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1339-1339 ◽  
Author(s):  
Kyu Lee Han ◽  
Cattlena M. Changpriroa ◽  
Harry L. Malech ◽  
Elizabeth M. Kang
Keyword(s):  
T Cells ◽  
Graft Versus Host Disease ◽  
Acute Gvhd ◽  
T Regulatory Cells ◽  
Regulatory Cells ◽  
Foxp3 Mrna ◽  
Host Disease ◽  
Graft Versus Host ◽  
A2ar Agonist

Abstract Abstract 1339 Poster Board I-361 Introduction Graft versus host disease (GVHD) remains a significant complication of allogenic stem cell transplantation and is a considerable cause of transplant morbidity and mortality. The recognition of the role of Foxp3+ regulatory T cells in immunomodulation has given rise to interest in using these cells to abrogate or modify the severity of graft versus host disease as well as in methods to increase their development during transplant. A less well known avenue of research is the targeting of the adenosine A2A receptor (A2AR). In ischemia models, activation of the Gs-coupled adenosine receptors play a role in terminating inflammation and improving survival of damaged and/or transplanted organs by directly down regulating the activity of the receptor bound T cell. We have shown previously that the use of a specific A2AR agonist known as ATL146e decreases the incidence and severity of GVHD as well as improves survival of mice in a GVHD transplant model (paper in submission). Methods In order to further understand the role of the agonist in GVHD abrogation we performed studies looking at the possible role of T regulatory cells in relation to the use of the agonist. Using a parental into irradiated F1 offspring transplant model (C57BL/6J [B6, H-2b] → B6D2F1/J [BDF1, H-2b/d]) we can induce GVHD manifested by weight loss and mortality in 100% of mice by infusing an additional 10 million donor T cells into mice previously engrafted with 10 million bone marrow donor cells and 800cGy of radiation. We administered the ATL146e or a PBS control by osmotic mini pumps resulting in continuous subcutaneous infusion for 14days starting one day before the donor T cell infusion. Mice that received only the congenic donor bone marrow transplant and no donor T cells did not develop GVHD and served as an additional control. Post transplant, splenocytes and peripheral blood cells were collected and stained for CD4, CD25 and FoxP3 and were analyzed by flow cytometry. The level of Foxp3 mRNA expression in lymphocytes was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and the concentration of IL-10 in serum was measured by enzyme-linked immunosorbent assay (ELISA). To identify the origin of activated T regulatory cells, we also performed transplants used a B6.PL-Thy1/CyJ (B6-Thy1.1, H-2b) mouse strain as the donor. Results From these studies we confirmed that the A2AR agonist, ATL146e, inhibited the weight loss and mortality associated with acute GVHD progression and seen in the PBS treated controls. More notably, treatment with ATL146e resulted in a 7 fold increase in CD4+CD25+FoxP3+ T regulatory cells in both the spleen and peripheral blood compared to our PBS treated group at days 14 to 20 after hematopoietic stem cell transplantation. From our Thy-1 disparate transplants we determined that the increased T regulatory cells were of donor origin. We also found that the expression of Foxp3 mRNA in splenocytes and the level of IL-10 in the serum was increased 3 fold and 2.4 fold respectively in the ATL146e treated mice. ATL146e agonist activity is very specific to A2AR. When using an alternate adenosine agonist less specific to A2AR, we did not see the same increases of Foxp3 or IL-10, nor did we see any decrease in the severity of the graft versus host disease. Conclusions Thus we believe that the specific activation of A2AR inhibits acute GVHD through the increase of donor-derived CD4+ CD25+ FoxP3+ immunosuppressive T regulatory cells. Our observation provides an additional mechanistic basis for the anti-inflammatory capacity of A2AR agonist in acute GVHD. Additional studies are ongoing to elucidate further the mechanism of the agonist's ability to increase the T regulatory population as well as the effects of combination therapies. Disclosures No relevant conflicts of interest to declare.

IL-27 Enhances Inducible Foxp3+ Treg Function to Prevent Acute Graft-Versus-Host Disease Lethality

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3343-3343
Author(s):  
Betty K. Hamilton ◽  
Jeongsu Do ◽  
Booki Min
Keyword(s):  
T Cells ◽  
Tumor Growth ◽  
Graft Versus Host Disease ◽  
Acute Gvhd ◽  
Ex Vivo ◽  
Foxp3 Expression ◽  
Graft Versus Host ◽  
Suppressive Function

Abstract Foxp3+ regulatory T cells (Tregs) are important regulators of graft-versus-host disease (GvHD) pathogenesis and a potential cellular therapy to prevent and treat GvHD. Thymus-derived (t)Tregs, while less frequent, are effective in preventing GvHD by ex vivo expansion, however Ag-non-specificity, stability, and cost-effectiveness are still critical issues to overcome. Peripherally-induced (p)Tregs are readily generated in large numbers in vitro;however, they are not suitable for therapeutic usage due to their rapid loss of Foxp3 expression and suppressive function when infused in the context of GvHD. The development of new approaches to increase in vivo stability/suppressive function of pTregs for clinical use is thus warranted. We recently demonstrated that IL-27 stimulation enhances suppressive function of Tregs (both tTregs and pTregs) in vitro and in vivo (Do, et al. Mucosal Immunol 2016). We hypothesized that IL-27 pre-stimulation would enhance pTreg function to suppress acute GvHD. Naïve CD4 T cells (from Foxp3GFP mice) were stimulated with anti-CD3/CD28 mAbs in the presence of TGFβ1 and IL-2 for 3 days, and >90% of the cells expressed GFP. Cells were then re-stimulated with the Abs for 3 more days with or without IL-27. IL-27 pre-stimulation enhanced Treg suppressive function in vitro, and was superior to that of rapamycin (Figure 1A). Lethally irradiated BALB/c mice received B6 BM cells with or without purified B6 CD3+ T cells. IL-27 pre-stimulated and control GFP+ pTregs were FACS sorted and transferred at the time of reconstitution. GvHD was evaluated by clinical features and survival. BM recipients that received T cells alone succumbed to death within 2 weeks of reconstitution due to lethal acute GvHD. While only ~40% of the control pTreg recipients were protected from lethal GvHD, all IL-27 pre-stimulated pTreg recipients were protected and survived (Figure 1B). CD4 and CD8 T cell expansion and ex vivo IFNγ production was substantially diminished with IL-27 pre-stimulated pTregs (Figure 1C). Next, lymphoma A20 cells transduced with the luciferase were transferred together with BM cells. Tumor growth was monitored. BM alone recipients had progression of tumor growth by day 14. Recipients of T cells alone died of lethal acute GVHD prior to day 14. Recipients of control and IL-27 stimulated pTregs had complete elimination of tumor cells. Of note, only 3 out of 5 recipients of control pTregs survived, with 2 succumbing to lethal GVHD prior to day 14 (Figure 1D). Although the total numbers of IL-27 pre-stimulated pTregs were higher than control pTregs, this was not statistically significant. GFP (Foxp3) expression of transferred pTregs, however, was markedly increased in IL-27 stimulated cells. We previously reported that IL-27 signaling in Tregs upregulates IL-10 and Lag3 (Do, et al. Mucosal Immunol 2016). We thus generated Il10-/- and Lag3-/- pTregs. Foxp3 expression of these pTregs was comparable to that of wild type pTregs, and IL-27 stimulation did not affect Foxp3 expression. IL-27-mediated enhanced in vitro suppression was lost in Lag3-/- but not in Il10-/- Tregs. Following IL-27 pre-stimulation, Il10-/- and Lag3-/- pTregs were transferred into recipients induced for acute GvHD. Foxp3 expression was similar at the time of transfer. IL-27 pre-stimulated Il10-/- pTregs protected recipients from acute GvHD lethality, however, Lag3-/- pTregs failed to protect from GVHD, even after IL-27 pre-stimulation (Figure 2A). IFNγ production of donor T cells was significantly reduced by IL-27 pre-stimulated Il10-/- but not by Lag3-/- pTregs (Figure 2B). In conclusion, we report that IL-27 pre-stimulation greatly enhances pTreg function, preventing acute GvHD lethality, while preserving a graft-versus-leukemia (GvL) effect. Lag3 on pTregs appears to be a key molecule mediating pTreg suppressive function enhanced by IL-27. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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