ALBERT

Description: Key Points There is a role for the posttranslational modification, neddylation, in regulation of immune responses mediated by dendritic cells. A role for neddylation in NF-κB signaling in dendritic cells was identified.

Description: The WT1 gene is a tumor-suppressor gene that was isolated as a gene responsible for Wilms' tumor, a childhood kidney neoplasm. We have previously reported that the WT1 gene is strongly expressed in leukemia cells with an increase in its expression levels at relapse and an inverse correlation between its expression levels and prognosis, thus making it a novel tumor marker for leukemic blast cells. Furthermore, WT1 antisense oligomers have been found to inhibit the growth of leukemic cells. These results strongly suggested the involvement of the WT1 gene in human leukemogenesis. The present study was performed to prove our hypothesis that the WT1 gene plays a key role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells, rather than a tumor-suppressor gene function. 32D cl3, an interleukin-3–dependent myeloid progenitor cell line, differentiates into mature neutrophils in response to granulocyte colony-stimulating factor (G-CSF). However, when transfected wild-type WT1 gene was constitutively expressed in 32D cl3, the cells stopped differentiating and continued to proliferate in response to G-CSF. As for signal transduction mediated by G-CSF receptor (G-CSFR), Stat3α was constitutively activated in wild-type WT1-infected 32D cl3 in response to G-CSF, whereas, in WT1-uninfected 32D cl3, activation of Stat3α was only transient. However, most interesting was the fact that G-CSF stimulation resulted in constitutive activation of Stat3β only in wild-type WT1-infected 32D cl3, but not in WT1-uninfected 32D cl3. Thus, WT1 expression constitutively activated both Stat3α and Stat3β. A transient activation of Stat1 was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation, but no difference in its activation was found. No activation of MAP kinase was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation. These results demonstrated that WT1 expression competed with the differentiation-inducing signal mediated by G-CSFR and constitutively activated Stat3, resulting in the blocking of differentiation and subsequent proliferation. Therefore, the data presented here support our hypothesis that the WT1 gene plays an essential role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells and represent the first demonstration of an important role of the WT1 gene in signal transduction in hematopoietic progenitor cells.

Description: In acute-type leukemia, no method for the prediction of relapse following allogeneic stem cell transplantation based on minimal residual disease (MRD) levels is established yet. In the present study, MRD in 72 cases of allogeneic transplantation for acute myeloid leukemia, acute lymphoid leukemia, and chronic myeloid leukemia (accelerated phase or blast crisis) was monitored frequently by quantitating the transcript of WT1 gene, a “panleukemic MRD marker,” using reverse transcriptase–polymerase chain reaction. Based on the negativity of expression of chimeric genes, the background level of WT1 transcripts in bone marrow following allogeneic transplantation was significantly decreased compared with the level in healthy volunteers. The probability of relapse occurring within 40 days significantly increased step-by-step according to the increase in WT1 expression level (100% for 1.0 × 10−2-5.0 × 10−2, 44.4% for 4.0 × 10−3-1.0 × 10−2, 10.2% for 4.0 × 10−4-4.0 × 10−3, and 0.8% for 〈 4.0 × 10−4) when WT1 level in K562 was defined as 1.0). WT1 levels in patients having relapse increased exponentially with a constant doubling time. The doubling time of theWT1 level in patients for whom the discontinuation of immunosuppressive agents or donor leukocyte infusion was effective was significantly longer than that for patients in whom it was not (P 〈 .05). No patients with a short doubling time of WT1 transcripts (〈 13 days) responded to these immunomodulation therapies. These findings strongly suggest that the WT1 assay is very useful for the prediction and management of relapse following allogeneic stem cell transplantation regardless of the presence of chimeric gene markers.

Description: Abstract 4707 Reduced-intensity stem cell transplantation (RIST) has come to be generally accepted as a method of allogeneic stem cell transplantation (SCT) for patients considered ineligible for myeloablative preparative regimens because of advanced age or comorbidities. We have recently reported unmanipulated nonmyeloablative HLA-haploidentical SCT using a conditioning treatment consisting of fludarabine, busulfan and anti-T-lymphocyte globulin and graft-versus-host disease (GVHD) prophylaxis consisting of tacrolimus and methylprednisolone (1 mg/kg) (Biol Blood Marrow Transplant 2006; 12:1073). In that study, the incidence of severe GVHD was only 10%. One of the mechanisms for such a low incidence of GVHD may have been caused by a reduced intensity of conditioning. Less intensive regimens should be associated with lower toxicity, a lower release of inflammatory cytokines, and potentially less GVHD; however, the mechanisms remain to be determined. Thus, using a murine MHC-haploidentical BMT model, BDF1(H-2b/d)®B6C3F1 (H-2b/k), that we established, we examined the influence of an intensity of conditioning treatment on GVHD. Recipient mice received T-cell-depleted bone marrow (5×106) and spleen cells (2×107) after total body irradiation (TBI) 13 Gy (myeloablative group) or 5 Gy (RIST group). Both groups of mice rapidly achieved donor engraftment. Recipients in the RIST group showed significantly fewer GVHD signs than those in the myeloablatve group. Histopathological examination of the myeloablative group on day 14 revealed various pathological changes in intestine (in particular large intestine) compatible to GVHD. In contrast, intestine samples from the RIST group showed few pathological changes with much less infiltration of donor T cells. Consequently, all recipients in the myeloablative group had died of GVHD by day 60, while all recipients survived for more than 3 months. These results clearly showed that the intensity of conditioning treatment influenced on the severity of GVHD and survival of recipients. Next, we investigated the mechanisms by which reduced intensity of conditioning ameliorated GVHD. Transplantation was performed using spleen cells that were labeled with the fluorescent cytoplasmic dye, carboxyfluorescein diacetate succinimidyl ester (CFSE), and cells in secondary lymphoid organs were analyzed by flow cytometry. The number of donor T cells in mesenteric lymph nodes on day 7 of the RIST group was significantly lower than that of the myeloablative group. In addition, a significantly increased number of host CD4+ T cells were recruited to secondary lymphoid organs on day 4 in the RIST group compared with the myeloablative group. An increased number of donor or host regulatory (Foxp3+CD4+) T cells were also observed in the RIST group. The levels of IFN gamma or IL-4 in lymphoid organs of the RIST group were higher than those of the myeloablative group. These results strongly suggest that host immune cells that survived conditioning treatment or cytokine milieu in secondary lymphoid organs contributed to the suppression of donor T cells during the initiation of GVHD. In addition, the expression of Th1 chemokine receptor, CXCR3, on donor T cells in secondary lymphoid organs and the expression levels of CXCL9, CXCL10, and CXCL11, ligands for CXCR3, in the large intestines were relatively lower in the RIST group, suggesting that the migration ability of donor T cells into GVHD target organs was negatively influenced by the intensity of conditioning. In conclusion, we showed that reduced intensity of conditioning improved the severity of GVHD, and that recipient immune cells, including regulatory T cells, together with reduced expression of inflammatory cytokines or chemokines, contributed to the improvement of GVHD in RIST. Disclosures: No relevant conflicts of interest to declare.

Description: ATG5 is a key protein that regulates autophagy, a vital cellular process whose role in various immune cells is poorly understood. A recent report showed that the deficiency of autophagy gene Atg16l1 in host DCs increased graft-vs-host disease (GVHD). Nevertheless, the direct role of autophagy in regulating T cell alloreactivity after bone marrow transplant (BMT) is unknown. In order to investigate the role of autophagy in T cells, we first analyzed the changes in autophagosome marker LC3 upon WT T cell activation. TCR stimulation with anti-CD3/CD28, increased cytosolic LC3-I and its membrane-bound LC3-II form. Interestingly, we found that the upregulation of LC3 was predominant in dividing cells, which lead us to hypothesize that autophagy is essential for T cell proliferation. Therefore we next explored if the deficiency in autophagy impaired T cell proliferation utilizing B6 T cell-specific ATG5 knockout (ATG5 KO T cell) mouse and hydroxychloroquine (CQ, a known inhibitor of autophagy). As hypothesized, when compared with WT controls, both CQ treated WT T cells and the ATG5 KO T cells, in vitro, demonstrated a significant decrease in proliferation as demonstrated by 3H-thymidine incorporation and CFSE staining (p

Description: Innate immune receptors like pattern recognition receptors (PRRs) including toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) like-receptors (NLR) on immune cells play an important role in initiating inflammatory responses to damage- and pathogen- associated molecular patterns (DAMPs and PAMPs) expressed on invading pathogens or released from damaged cells. Although it is well known that DAMPs directly modulate innate immune functions, it is less clear whether DAMPs directly regulate T cell intrinsic function. Members of the sialic acid binding Ig-like lectin (Siglec) family have immunoreceptor tyrosine-based inhibitory motifs (ITIM) or ITIM-like regions in their intracellular domain that negatively regulate immune activation induced by DAMPs. Our previous data suggested that the Siglec- G-CD24 interaction in host APCs plays an important role in the negative regulation of graft-versus host (GVH) responses. However, the T cell autonomous role of Siglec-G in the regulation of T cell responses is not known. Because Siglecs are important negative regulators of immune responses, we tested the hypothesis that the deficiency of Siglec-G in donor T cells would enhance GVHD. To test our hypothesis, we first examined detailed phenotypic analysis of various T cell subsets and activation markers in naïve Siglec-G-/- and wild-type (WT) B6 animals and found similar distribution of naïve, memory, effector and regulatory T cells. In order to examine whether the absence of Siglec-G in donors affects GVHD, WT-BALB/cmice were lethally irradiated (850cGy) and transplanted on day 0 with 5x106 bone marrow and 0.5x106 splenic CD90+ T cells from either syngeneic WT-BALB/c, allogeneic MHC-mismatched WT-B6 or Siglec-G-/- animals. The recipients receiving donor T cells from Siglec-G-/- animals showed a significantly worse survival compared to allogeneic WT-B6 animals (p

Description: NLRP6 (NOD-like receptor family pyrin domain containing 6) is an important inflammasome component and is highly expressed in intestinal epithelial and in immune cells. NLRP6 mediated inflammasome activation plays a critical role in response to intestinal infection and preventing dysbiosis of gut microbiota through the secretion of IL-18 and mucus. However, we recently found that NLRP6 plays a pathogenic role in GVHD that is independent of microbial dysbiosis, which is in contrast to its well-appreciated microbiome-dependent protective role in intestinal colitis and tumorigenesis. Interestingly, we also found that activated T cells increased NLRP6 expression, but the T cell autonomousrole of NLRP6 in regulating T cell responses is unknown. Because NLRP6 is an important regulator of GVH responses, we tested the hypothesis that NLRP6 deficiency in donor T cells would ameliorate GVHD. To test our hypothesis, we first performed adetailed phenotypic analysis of various T cell subsets and activation markers in naïve NLRP6-/-and wild-type (WT) B6 animals and found a similar distribution of naïve, memory, effector and regulatory T cells. In order to examine whether the absence of NLRP6 in donors affects GVHD, WT-BALB/canimals were lethally irradiated (700cGy) and transplanted on day 0 with 5x106bone marrow and 1.0x106 splenic CD90+T cells from either syngeneic WT-BALB/c, allogeneic MHC-mismatched WT-B6 or NLRP6-/-animals. Contrary to our hypothesis, the recipients receiving donor T cells from NLRP6-/-animals showed a significantly worse survival compared to allogeneic WT-B6 animals (p

Description: Abstract 4117 Related haploidentical donors, as cord blood, can be alternative donor sources in stem cell transplantation (SCT). Severe GVHD, however, has interfered the progress of haploidentical SCT (haploSCT). To deal with this strong GVHD, T cell depletion has usually been used in US and European countries. In order to pursue the controllable GVL effect by T cells, we have performed unmanipulated haploSCT using myeloablative or reduced intensity preconditioning regimen accompanied with intensified GVHD prophylaxis, including steroids. In this meeting, we will summarize our experience of haploSCT for more than ten years. From August 1998 to September 2010, we have performed 351 cases of haploSCT (all cases were HLA 2–3 antigen mismatched in GVH direction). Patients' characteristics are sex: male 186, female 168, age: 16–65 years old (median 39), disease: AML/MDS 149, ALL 81, ML 67, others 54. Eighty-three percent of cases underwent SCT in non-complete remission (non-CR) status. Patients under 45 years old underwent myeloablative preconditioning regimen consisting of FLU/CA/CY/TBI 8Gy (haplo-full, n=100), and patients over 45 years old or with comorbidities or repetitive SCT (including second to fifth SCT) underwent reduced intensity preconditioning regimen consisting of FLU/(CA)/BU/ATG or FLU/(CA)/MEL/ATG (haplo-mini, n=251). High dose Ara-C (CA) was optional to reduce tumor burden. As ATG, ATG (Fresenius) 8mg/kg, or thymoglubulin (genzyme) 2–4mg/kg were integrated into conditioning treatments mainly for reduced-intensity transplantation. GVHD prophylaxis consisted of taclolimus (TAC), methylprednisolone (mPSL) 2mg/kg/day, short term MTX, and mycophenolate mofetil (MMF) 15mg/kg/day in haplo-full, and TAC, and mPSL 1mg/kg/day in haplo-mini, respectively. For elderly patients over 50 years old in haplo-mini, MMF was added. Hematopoietic engraftment in haploSCT was as rapid as that in HLA-identical SCT, except 10 cases of graft rejection. The median time to reach a neutrophil account of 〉0.5 × 109/l was 10 days for haplo-mini and 13 days for haplo-full. Platelet recovery was achieved in 66 % and 60% of patients undergoing haplo-mini and haplo-full, respectively. The median time to reach a nontransfused platelet count of 3 20 × 109/l was 22 days for haplo-mini and 33 days for haplo-full. Sixty percent of haplo-mini patients and 54 % of haplo-full patients did not develop acute GVHD. Acute GVHD (grade II-IV) was observed in 20% for haplo-mini and 36 % for haplo-full. Overall survival at five years was 30% for haplo-full and 40% for haplo-mini, respectively. If limited to CR cases, overall survival reached over 60% in haplo-mini. There is no difference in survival rate among patients' diseases. In multivariate analysis on survival using variables, including disease status before transplantation, haplo-full vs haplo-mini, mismatches in GVH direction, mismatches in HVG direction, patients' age, and the number of transplantation times, the disease status (CR) was found to be only a significantly favorable factor (P= 0.0026). Unmanipulated haploSCT is feasible and effective for refractory diseases. ATG dose used in haplo-mini is critical, and rather low compared with that of European cases reported so far. Although it should be too early to refer long term outcome, unmanipulated haploSCT could be considered as an option to control refractory diseases. Disclosures: No relevant conflicts of interest to declare.

Description: Intestinal epithelial cells play an important role in the maintenance of host microbiota as well as intestinal homeostasis by expression of pattern recognition receptors (PRRs). NLRP6 (NOD-like receptor family pyrin domain containing 6) is an important inflammasome component and is highly expressed on the intestinal epithelium and in immune cells. NLRP6 mediated inflammasome activation plays a critical role in intestinal infection and in preventing dysbiosis of gut normal microbiota thorough the secretion of IL-18 and mucus. In addition, NLRP6 knock out (NLRP6-/-) mice show greater severity and mortality of inflammatory bowel diseases (IBD). Given its unique functions in GI tract and in immune cells, we determined its role in the severity of GI GVHD by testing the hypothesis that the absence of the NLRP6 in hosts would enhance GVHD. We utilized the well-characterized MHC disparate BALB/c→B6 model of GVHD. B6 wild type (WT) and B6 NLRP6-/- animals were lethally irradiated (10Gy) and transplanted with 5x106 BM and 3x106 splenic T-cells from either syngeneic WT-B6 or allogeneic BALB/c donors. Contrary to the hypothesis and in contrast to IBD model, allogeneic B6 NLRP6-/- animals showed significantly improved survival compared to the allo-B6 WT animals (p