ALBERT

Description: Allogeneic hematopoietic cell transplantation (HCT) is an effective treatment for a variety of hematologic malignancies. However, overall success of HCT is limited due to the immunologic recognition and destruction of host tissues resulting in graft-versus-host-disease (GvHD). In GvHD, donor derived immune cells recognize and destroy recipient epithelial tissues; predominantly liver, gastrointestinal (GI) mucosa, and skin. It has been reported that GI toxicity and crypt loss is a strong predictor of GvHD morbidity and mortality. Therefore, GI protective strategies may diminish GvHD severity. R-spondin1 (Rspo1) is an epithelial mitogen that stimulates mucosal growth in both small and large intestines. Therapeutic administration of Rspo1 has been shown to reduce the loss of body weight, diarrhea and rectal bleeding in experimental mouse colitis models. Histological evaluation revealed that Rspo1 improved mucosal integrity by stimulating crypt cell growth and mucosal regeneration in colitis-induced mice. Because the symptoms and pathogenesis of GI-GvHD are similar to colitis models, we chose to examine the impact of Rspo1 on mice with GvHD. Conventional CD4+/CD8+ T cells (Tconv) isolated from FVB (H-2q) animals were transplanted along with T cell-depleted bone marrow (TCD-BM) into lethally irradiated BALB/c (H-2d) mice. Mice received either 100μg Rspo1 protein in 100μL PBS or 100μL PBS alone IV from day 1 to day 6 post-transplantation. Surprisingly, mice receiving Rspo1 had significantly increased rates of mortality. Mice receiving Rspo1 experienced 100% mortality by day 6 post-HCT. In contrast, mice receiving only PBS demonstrated 0% mortality at 20 days post-HCT. Mortality was not a result of Rspo1 toxicity, as no morbidity or mortality was observed in the absence of allogenic Tconv in mice transplanted with either syngenic HCT or allogeneic TCD-BM alone. In order to assess whether the higher mortality experienced by Rspo1 treated mice was due to an explosive proliferation of Tconv, the transplant experiment was repeated with donor Tconv isolated from luciferase transgenic (luc+) H-2q mice. Groups receiving Rspo1 versus PBS did not show significantly different bioluminescence imaging signal intensity, suggesting that rapid proliferation of donor Tconv was not the basis for the observed differences in mortality. The overall mouse mortality in this experiment was consistent with the previous experiment. In an attempt to uncover the mechanism, we found no differences in the Perforin-Granzyme B or Fas/Fas ligand expression between control and treatment groups. Cytokine milieu (IFNγ, TNFα, IL-17, and IL-2) assessment in transplanted mice did not show significant differences in cytokine profiles of control versus Rspo1 treatment. Mouse autopsy demonstrated that transplanted mice receiving Rspo1 had marked crypt hyperplasia, apoptotic cells, and increased mitoses when compared PBS recipients. All other organ systems were considered normal in both groups by a veterinary pathologist. Blood cultures of both groups were negative for bacteria. CBC, liver enzymes, and electrolyte panels were normal in both groups. In order to assess lethal irradiation as a contributing factor to the increased mortality of Rspo1 treated mice, we repeated the above transplant experiment including non-irradiated immunodeficient Rag2−/−, γ-chain−/− H-2d recipients in addition to the lethally irradiated H-2 d recipients. Our mortality findings in the lethally irradiated group were consistent with the above mentioned transplant experiments showing rapid mortality rates in recipient mice receiving Rspo1. However, non-irradiated Rag2−/− γ-chain−/− mice that received Rspo1 had almost identical mortality rates to those receiving PBS; all mice surviving over 20 days post HCT. This finding underlines the critical importance of lethal irradiation as a contributing factor in Rspo1 treatment mortality. These findings suggest that the beneficial effects of Rspo1 in the colitis model may not be reproducible in GvHD. Furthermore, our studies indicate that the lethal effects of Rspo1 may be due to a myriad of factors including allogenic mismatch and lethal irradiation.

Description: Allografting as a curative approach for many hematological malignancies is hampered by the occurrence of acute graft-versus-host disease (aGvHD). Interleukin (IL)-18 stimulates T helper 1 (Th1) and Th2-mediated immune responses and has been shown to modulate aGvHD. It is still unknown whether increased IL-18 levels during aGvHD are of host or donor origin and how the absence of IL-18 impacts migration and expansion of conventional CD4+CD25− (Tconv) and CD4+CD25+ regulatory (Treg) T cells in vivo. By utilizing IL-18 gene deficient donor versus recipient animals we found that the major cytokine production during the early phase of aGVHD induction was recipient derived, while donor hematopoietic cells contributed significantly less. By generating IL-18−/ − luciferase transgenic mice we were able to investigate the impact of IL-18 on Tconv and Treg expansion and trafficking with in vivo bioluminescence imaging. While migration to secondary lymphoid organs was not significantly impacted by the absence of host IL-18, Tconv but not Treg expansion increased significantly. Absence of host IL-18 production translated into lower IFN-γ levels in the early phase after transplantation. We conclude that host derived IL-18 is a major factor for IFN-γ production that may have a protective effect on CD4+ mediated aGvHD, but is non-essential for Treg expansion in an allogeneic environment.

Description: CD4+CD25+ regulatory T cells (Treg) have been demonstrated to reduce the severity of acute graft-versus-host disease (aGvHD) in murine models of bone marrow transplantation. However, the surface molecules that are critical for suppression are unclear. The TNF-R superfamily member CD30 has been shown to be expressed on regulatory T cells that down-modulate nickel specific immune responses and to be relevant for Treg mediated protection from allograft rejection. Deficiency of the CD30 molecule (CD30−/−) is associated with impaired thymic negative selection and augmented T cell autoreactivity. Therefore, we investigated the role of CD30 signaling in Treg function in an aGvHD model. Treg derived from CD30−/ − animals were significantly less effective in preventing aGvHD lethality (wt vs. CD30−/ − p=0.002). Signal intensity derived from expanding luciferase expressing alloreactive conventional T cells (Tconv) was significantly higher if CD30−/ − Treg as compared to wt Treg (p=0.007) were transferred as assessed by bioluminescencent based imaging. Blockade of the CD30/CD153 pathway with a neutralizing anti-CD153 mAb during the early (days −2 to +4) but not late (days +4 to +10) phase of adoptive Treg transfer reduced Treg mediated protection from proinflammatory cytokine accumulation and apoptosis of donor-type CD4 and CD8 T cells. In vivo bioluminescence imaging demonstrated intact Treg homing, but reduced expansion when CD153 was blocked during the early phase after adoptive transfer. CD30 surface expression on Treg increased with alloantigen exposure and CD153 expression on recipient-type dendritic cells increased in the presence of an irradiation induced proinflammatory environment but not when T cell depleted bone marrow and Tconv were transferred into non-irradiated Rag 2−/ −γc−/ − recipients. These data are the first to demonstrate that early CD30 signaling is relevant for Treg mediated aGvHD protection after major MHC mismatch bone marrow transplantation.

Description: Based on their ability to control T-cell homeostasis, Foxp3+CD4+CD25+ regulatory T cells (Tregs) are being considered for treatment of autoimmune disorders and acute graft-versus-host disease (aGVHD). When combining Tregs with the immunosuppressant rapamycin (RAPA), we observed reduced alloreactive conventional T-cell (Tconv) expansion and aGVHD lethality compared with each treatment alone. This synergistic in vivo protection was paralleled by intact expansion of polyclonal Tregs with conserved high FoxP3 expression. In contrast to Tconv, activation of Tregs with alloantigen and interleukin-2 preferentially led to signal transducer and activator of transcription 5 (STAT5) phosphorylation and not phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway activity. Expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a negative regulator of the PI3K/Akt/mTOR pathway, remained high in Tregs but not Tconv during stimulation. Conversely, targeted deletion of PTEN increased susceptibility of Tregs to mTOR inhibition by RAPA. Differential impact of RAPA as a result of reduced usage of the mTOR pathway in Tregs compared with conventional T cells explains the synergistic effect of RAPA and Tregs in aGVHD protection, which has important implications for clinical trials using Tregs.

Description: FoxP3+CD4+CD25+ regulatory T-cells (Treg) have been shown to effectively reduce the severity of experimental acute graft-versus-host disease (aGvHD) while sparing graft-versus-leukemia activity. These findings, in concert with the observation that human and murine Treg share functional characteristics, have fueled interest in clinical trials to control aGvHD. Recent data indicates that the immunosuppressant rapamycin (RAPA) in contrast to cyclosporine A does not interfere with in vivo function of Treg and could enhance Treg expansion in vitro by a yet unknown mechanism. To investigate the impact of mTOR inhibition on proliferating Treg and Tconv, both cell types were exposed to CD3/CD28 Mabs in the presence of different RAPA concentrations in vitro. Phosphorylation of mTOR downstream products p70S6K1 and 4E-BP1 were assessed by western blot and flow cytometry. Inhibition of the phosphorylation of p70S6K1 and 4E-BP1 was observed in both populations in the presence of RAPA. Interestingly, Treg were more resistant to mTOR inhibition as compared to Tconv and displayed significantly higher phosphorylated products in the presence of RAPA at 10 nM (MFI Treg vs Tconv, p