Acute Graft-versus-host Disease In Murine Model Research Articles

Reconstitution of IL-17-Producing T Helper Cells After Allogeneic Hematopoietic Cell Transplantation

AbstractAbstract 4167Acute graft-versus-host disease (GvHD) following allogeneic hematopoietic cell transplantation (HCT) has been classically assumed to be mediated by T helper cells type 1 (Th1), characterized by the production of interferon-γ (IFN-γ). Recently, Interleukin 17A (IL-17)-producing CD4+ T helper type 17 (Th17) cells have also drawn attention as possible effector cells of acute GvHD in murine models. Their role following allogeneic HCT in humans is unknown. We hypothesized that IFN-γ/IL-17-production and quantity of T helper cells might depend on the time-point after HCT, immune responses to allo-antigens (GvHD) or pathogens (e.g. bacterial infection, CMV reactivation) and the presence of T cell depleting antibodies (e.g. ATG). To explore this hypothesiswe initiated a prospective study to investigate the reconstitution of Th1, Th1/17 and Th17 cells in patients after HCT.80 consecutive patients with various hematologic disorders undergoing allogeneic human leukocyte antigen (HLA)-matched HCT at our center between 12/2009 and 9/2010 were included into the study. Blood samples were collected once in the 1st month, 2nd month and 3rd month after HCT. To quantify IL-17- and IFN-γ-producing T helper cells, we used surface staining for CD3 and CD4 followed by intracellular cytokine staining for IFN-γ and IL-17 in PBMCs. T helper cells producing both IFN-γ and IL-17 (IFN-γ+IL-17+) were termed Th1/17 cells, T helper cells producing only either cytokine alone are indicated as Th17 cells (IFN-γ−IL-17+) or Th1 cells (IFN-γ+IL-17−). For each time period patient cohorts were defined according to the subsequent criteria: (i) bacterial infection (C-reactive protein >50 mg/L, positive blood culture and/or fever in the absence of viral or fungal infection), (ii) CMV reactivation (positive CMV-specific PCR), (iii) acute GvHD (according to the Seattle criteria) and (iv) ATG in the conditioning regimen (dose of 20mg/kg on day -3, -2 and -1 before HCT). As a reference group we chose time-matched and age-matched patients that did not meet any of the criteria under investigation. Student′s t test (two sided, unpaired) was used for statistical evaluation.In all patients with no relevant complication (absence of bacterial infection, CMV reactivation, acute GvHD) and no ATG in the conditioning regimen Th1, Th1/17 and Th17 cells were detectable within the first month after HCT. However, these T helper cell subsets did not reconstitute to levels of healthy controls within the first 3 month after HCT. In contrast to Th1 cells, no further expansion of Th1/17 and Th17 cells was observed following the 1st month after HCT. ATG during conditioning significantly reduced the frequency of Th17 cells at all time-points analyzed (median decrease: 1st month, 71.5%, P=0.0049; 2nd month, 82.5%, P=0.0002; late engraftment, 71.4%, P=0.0011). Th1/17 cells were also suppressed in patients with ATG, although this reduction was less prominent and reached no significance following the 2nd month after HCT (median decrease: 1st month, 76.19%, P=0.012; 2nd month, 70.11%, P=0.054; late engraftment, 50.7%, P=0.69). Finally, Th1 cells were not significantly reduced in patients receiving ATG compared to time-matches controls (median decrease: 1st month: 89.18%, P=0.34; 2nd month: 62.7%, P=0.21; late engraftment: 19.9%, P=0.8), indicating that the suppressive effect of ATG is less pronounced on Th1 and TH1/17 cells, compared to Th17 cells. Acute GvHD°I was not associated with significant changes in the size of the Th1, TH1/17 or Th17 cell subsets. In patients with GvHD°II-IV a tendency towards increased counts of Th1, TH1/17 and TH17 cells in the peripheral blood was observed. However, these changes were not statistically different compared to time-matched controls. CMV reactivation triggered the expansion of all T helper subsets and Th1 cells showed the strongest increase (median increase: Th1, 449.1%, P=0.00075; Th17, 74.9%, P=0.00069; Th1/17, 97.1%, P=0.00012). In contrast, no significant changes were found in the T helper cell compartment of patients with bacterial infection compared to time matched controls.In conclusion, quantitative reconstitution of Th1, Th1/17 and Th17 cells is impaired within the first 3 months after HCT, especially when ATG is administered during conditioning. CMV reactivation, but not bacterial infection, triggered the absolute expansion of these T cell subsets. Disclosures:No relevant conflicts of interest to declare.

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

Abstract 2987Acute 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.

Association of Serum Interleukin-7 Levels With the Development of Acute Graft-Versus-Host Disease

Morbidity from acute graft-versus-host disease (GVHD) limits the success of allogeneic hematopoietic stem-cell transplantation (HSCT) to treat malignancy. Interleukin-7 (IL-7), the principal homeostatic cytokine for T cells, is required for acute GVHD in murine models. In contrast to inflammatory cytokines (eg, IL-2, tumor necrosis factor alpha), IL-7 has not been studied extensively in the clinical transplant setting relative to its relationship with acute GVHD. We evaluated the association of serum IL-7 levels with acute GVHD in 31 patients who were uniformly treated in a prospective clinical trial with reduced-intensity allogeneic HSCT from human leukocyte antigen-identical siblings. GVHD prophylaxis consisted of cyclosporine and methotrexate. Serum IL-7 levels and lymphocyte populations were determined at enrollment, the day of transplantation before the allograft infusion, and at specified intervals through 12 months post-transplantation. As expected, IL-7 levels were inversely correlated with T-cell populations (P < .00001). Acute GVHD was significantly associated with higher IL-7 levels at day +7 (P = .01) and day +14 (P = .00003) post-transplantation as well as with the allograft CD34(+) cell dose (P = .01). IL-7 levels at day +14 also correlated with the severity of acute GVHD (P < .0001). In logistic regression models, these factors were highly sensitive (up to 86%) and specific (100%) for classifying whether patients developed acute GVHD. These data support preclinical observations that IL-7 plays a critical role in inducing acute GVHD and provide a rational basis for novel approaches to prevent and treat acute GVHD through modulation of the IL-7 pathway.

Granulocyte Colony-Stimulating Factor–Mobilized Allogeneic Stem Cell Transplantation Maintains Graft-Versus-Leukemia Effects Through a Perforin-Dependent Pathway While Preventing Graft-Versus-Host Disease

Minimization of graft-versus-host disease (GVHD) with preservation of the graft-versus-leukemia (GVL) effect is a crucial step to improve the overall survival of allogeneic bone marrow transplantation (BMT) for patients with hematological malignancies. We and other investigators have shown that granulocyte colony-stimulating factor (G-CSF)–mobilized allogeneic peripheral stem cell transplantation (PBSCT) reduces the severity of acute GVHD in murine models. In this study, we investigated whether G-CSF–mobilized PBSC maintain their GVL effect in a murine allogeneic transplant model (B6 → B6D2F1). B6 mice (H-2b) were injected subcutaneously with human G-CSF (100 μg/kg/d) for 6 days and their splenocytes were harvested on day 7 as a source of PBSC. G-CSF mobilization dramatically improved transplant survival compared with nonmobilized controls (95% v0%, P &lt; .001). Systemic levels of lipopolysaccharide and tumor necrosis factor- were markedly reduced in recipients of allogeneic G-CSF–mobilized donors, but cytolytic T lymphocyte (CTL) activity against host tumor target cells p815 was retained in those recipients. When leukemia was induced in recipients by coinjection of p815 tumor cells (H-2d) at the time of transplantation, all surviving recipients of G-CSF–mobilized B6 donors were leukemia-free at day 70 after transplant, whereas all mice who received T-cell–depleted (TCD) splenocytes from G-CSF–mobilized B6 donors died of leukemia. When splenocytes from G-CSF–mobilized perforin-deficient (pfp−/−) mice were used for transplantation, 90% of recipients died of leukemia, demonstrating that perforin is a crucial pathway mediating GVL effects after G-CSF–mobilized PBSCT. These data illustrate that G-CSF–mobilized allogeneic PBSCT separate GVL from GVHD by preserving perforin-dependent donor CTL activity while reducing systemic inflammation.