ALBERT

Description: Originally mast cells have been known as effector cells in the IgE-mediated allergic responses. In addition, recent studies demonstrated that mast cells exerted pro-inflammatory or anti-inflammatory effects in complicated immune response depending on the situation. In allogeneic hematopoietic stem cell transplantation (HSCT), exact role of mast cells in graft-versus-host disease (GVHD) remains unclear. First we investigated whether host mast cells protect acute GVHD or not in a murine HSCT model using mast cell deficient mice. When lethally irradiated wild type (WT) B6 or mast cell deficient mice were transplanted with bone marrow cells and spleen cells from WT Balb/c mice, mast cell deficient B6 host mice showed significantly lower survival rate than WT B6 mice (p

Description: Abstract 3719 Cytokine-induced killer (CIK) cells are ex vivo–expanded T lymphocytes expressing both natural killer (NK)– and T-cell markers. We have reported that adoptive transfer of allogeneic CIK cells in a murine model caused minimal graft versus host disease (GVHD) with retention of antitumor activity mediated by NKG2D, which is an activating receptor expressed on NK cells. With the purpose of potential application of CIK cells in a clinical hematopoietic stem cell transplantation, the problem we have to consider next is whether CIK cells could promote an engraftment and facilitate an immune reconstitution. To this end, lethally irradiated BALB/c mice were injected with minimal number of MHC incompatible C57BL/6 bone marrow (BM) cells alone, in which almost half of mice died because of graft failure, or with CIK concurrently. The mice receiving BM plus CIK cells survived with 93% without GVHD, demonstrating that CIK cells significantly promote an engraftment (P

Description: Abstract 2777 We have reported that bone marrow derived dendritic cells with psoralen and UVA (PUVA-DCs) treatment acquired tolerogenicity in mice. With the purpose of potential application of PUVA-DCs in a clinical hematopoietic stem cell transplantations (HSCT) for graft-versus-host disease (GVHD), we showed that mixed lymphocyte reaction (MLR) was strongly inhibited when PUVA-DCs from the stimulator strain were added to the coculture (Stimulator (S): conventional DCs obtained from C57BL/6, Responder (R): splenocytes obtained from Balb/c, PUVA-DCs: C57BL/6). This suggests that infusion of host-typed PUVA-DCs would become a novel therapeutic approach for GVHD. However utilizing host-typed DCs has problems because of leukemic cell contaminations or low efficiency of cell culture from the patients receiving repetitive chemotherapy. Therefore next concern is whether PUVA-DCs generated from BM donor or even strangers would have same tolerogenicity as host-typed PUVA-DCs do. To test this, we performed MLR by adding PUVA-DC generated from the same strain of responder or third party strain (S: conventional DCs obtained from C57BL/6, R: splenocytes obtained from Balb/c, PUVA-DC: C57BL/6 or C3H). Proliferation was significantly inhibited when PUVA-DC generated from the stimulator strain were added to the coculture (p

Description: Cell adoptive immunotherapy protect from lethal graft-versus-host disease (GVHD) while preserving graft-versus-tumor effects is ideal in allogeneic hematopoietic stem cell transplantation (HSCT) in patients with hematologic malignancies. Moreover, the use of third party cells would open a huge source of availability and feasibility across major histocompatibility barriers. In fact, some studies demonstrated that infusion of third party derived cells such as whole bone marrow cells or iNKT cells could ameliorate lethal GVHD in a murine model. Cytokine-induced killer (CIK) cells are ex vivo-expanded T lymphocytes expressing both natural killer and T-cell markers. We have previously reported that donor-type CIK cells have potential to separate graft-versus-tumor effects from GVHD by eliminating host dendritic cells due to the enhanced killing activity by interferon-gamma. Actually, donor-typed CIKs infusion for refractory hematological malignancies after HSCT has been started in some clinical trails. In the present study, we examine the effect of third party cells against GVHD protection, in particular third party CIK cells for prevention from lethal GVHD in a murine model of allogeneic HSCT. To test this, lethally irradiated host Balb/c (H-2d) mice were given C3H (H-2k) or B6 (H-2b) bone marrow cells and splenocytes to induce lethal GVHD with/without third-party cells (C57/BL6 or C3H) such as CIK cells, whole splenocytes, or bone marrow derived dendritic cells (BMDCs) on day 0, which were cultured from bone marrow cells with GM-CSF. Mice receiving third party CIK cells showed much less GVHD and significant survival benefit compared those with third party splenocytes, or BMDCs as shown in the figure below. Interestingly, when infusion of third party splenocytes was delayed until day 4 after bone marrow transplantation, host mice receiving splenocytes survived much better compared with mice receiving splenocytes on day 0, even though reduced number of third party splenocytes was used. This result indicated that timing of third party cell infusion was quite important, especially when third party splenocytes were used. As expected, the mice given reduced number of third CIK cells also showed excellent survival with much less GVHD. Taken together, our results clearly demonstrated that third party CIK cells have strong potential to prevent lethal GVHD without attention to timing of cell infusion. Next, to further investigate the advantage of third party CIK cells rather than whole splenocytes, we compared the hematological reconstitution between the mice given third party CIK cells on day 4 after BMT and whole splenocytes. Both mice showed much less GVHD with the same level, however the recovery of white blood cell count on day 21 after BMT was significantly better in the mice receiving third party CIK cells. In conclusion, infusion of third party CIK cells has strong potential to prevent lethal GVHD with faster hematological recovery after HSCT. Disclosures No relevant conflicts of interest to declare.

Description: Background and Objective Rate of leukemic invasion to kidney varied among reports, and because of it, the clinical significance of renal involvement in pediatric acute lymphoblastic leukemia at diagnosis has remained controversial. Conventionally, renal involvement have been diagnosed by ultrasonography according to the definition of unilateral or bilateral nephromegaly based on the length of major or minor axis 〉2SD, diffuse abnormal findings, or mass legions. However to detect the leukemic involvement much more accurately, we utilized contrast-enhanced CT to evaluate renal mass legions and to measure renal volume by utilizing 3D reconstruction models of CT images. Leukemic involvement in the kidney was defined only when markedly decrease of renal volume before and after induction chemotherapy and/or mass lesions were observed. The aim of this study was to assess whether our 3D-CT-based criteria determined by utilizing the modern medical technology could be superior to the conventional criteria based on ultrasonography and to reveal significant clinical impact of renal involvement. Materials and Methods From 2006 to 2012, 23 children were diagnosed with acute lymphoblastic leukemia in our hospital. Enhanced CT scan was performed to the all patients before induction therapy, and a follow-up CT scan was performed after induction therapy or later stages on the patients who had abnormal CT findings at diagnosis. Mass legions, length of major and minor axis, and volume changes by utilizing enhanced CT and 3D reconstruction models calculated with the software SYNAPSE VINCENT (FUJIFILM MEDICAL), were evaluated. Results 　15 out of 23 patients (65.2%) presented with renal involvement when conventional criteria was used (major or minor axis 〉2SD, or diffuse abnormal findings or mass legions). However when applied to our 3D-CT- based criteria (mass legions or marked decrease of renal volume before and after induction therapy), only 6 patients out of 23 patients (26.1%) showed renal involvement (volume change alone 1, mass lesions alone 3, mass lesion with volume change 2). Quite big difference of renal involvement between criteria was due to the patients who fulfilled conventional criteria did not show the volume decrease of kidney before and after induction chemotherapy, even though these patients reached complete remission in bone marrow and markedly decrease of spleen volume measured by our 3D-CT methods. It suggested that these cases did not have true renal involvement and that the nephromegaly defined by only renal length included many false positive cases. Most importantly, renal mass legions could be detected only by enhanced CT, not by ultrasonography in some cases. Frequency of the presence of central nervous system and extramedullary involvement except lymphadenopathy were significantly higher in the patients with renal mass legions (p=0.005 and P=0.043, respectively). Moreover, the patients with renal mass legions had tendency to relapse. Conclusion Our 3D-CT-based criteria for detection of leukemia invasion to kidney is much more reliable than the conventional criteria based on nephromegaly measured by major or minor axis. Renal mass legions strongly correlated with central nervous system and extramedullary involvement except lymphadenopathy. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3011 Cytokine-induced killer (CIK) cells are ex vivo-expanded T lymphocytes expressing both natural killer (NK)- and T-cell markers. We have reported that adoptive transfer of allogeneic CIK cells in a murine model caused minimal graft-versus-host disease (GVHD) with retention of antitumor activity mediated by NKG2D, which is an activating receptor expressed on NK cells. The mechanism of suppression of GVHD with CIK cells is not fully understood. Host residual dendritic cells (DCs) are most important cells in initiating GVHD reaction. Therefore, we hypothesized that alloreactive NK cells, even when infused in large numbers, do not cause GVHD by killing recipient DCs with a same mechanism as CIK cells kill tumor cells. To test this, DCs generated from rodent bone marrow cells were used for 51-Cr release cytotoxicity assays as target cells. We demonstrated that though autologous CIK cells (Balb/c) had relatively strong killing activity against mature DCs (Balb/c), allogeneic CIK cells (B6) had much more killing activity even from at a 10:1 effector -target ratio as shown in Fig 1 below. In addition, killing activity against DCs did not changed with/without adding NKG2D blocking antibody, suggesting that there were possible mechanisms for cell killing other than NKG2D/NKG2D ligand system in CIK cells. To further evaluate whether allogeneic CIK cells eliminate host DCs to reduce GVHD in vivo, lethally irradiated Balb/c recipients were given BM (B6) with CIK cells or splenocytes to compare the number and activation status of residual host-typed DCs in the spleen after bone marrow transplantation. As shown in Fig 2, on day 1 after BMT absolute number of host DCs in spleen receiving splenocytes and CIK cells were almost same, however, the numbers of DCs in the mice receiving BM alone were much less. On day 3, absolute number of host DCs in all groups decreased. In particular, the number of host DCs in the mice receiving CIK cells dramatically decreased and finally reached to the same number of host DCs in the mice receiving BM alone. On the contrary, the number of host DCs in the mice receiving splenocytes didn't decrease so much and was significantly greater than number of host DCs in the mice receiving BM alone. Considering the above, when allo-CIK cells first met to DCs, both DCs and CIK cells were expanded on day 1 after BMT, and then allo-CIK cells turned to kill host DCs. In conclusion, allogeneic CIK cells caused less GVHD due in part to elimination of host DCs. Disclosures: No relevant conflicts of interest to declare.

Description: Background Thymic FDG uptake due to rebound thymic hyperplasia after chemotherapy for cancers including malignant lymphoma (ML) is relatively widely-recognized phenomenon, especially in children. However, its occurrence rate based on ages and intensity of chemotherapy for various cancers has not really investigated. Therefore, to make clear distinction between thymic hyperplasia and lymphoma recurrence would be often difficult. To avoid misdiagnosis or unnecessary biopsies, recent report emphasized the importance to be aware of the possibility of thymic hyperplasia after chemotherapy. Another group suggested that SUV alone could be sufficient for differential diagnosis. However the duration showing rebound thymic hyperplasia after chemotherapy has never been studied so far, and there are no reports about the difference between rebound thymic hyperplasia and malignancy from the aspect of the morphological thymic uptake pattern. In this study, we investigated: 1) how long rebound thymic hyperplasia could last and 2) whether SUV alone would be sufficient or using additional findings such as the pattern of uptake (diffuse or nodular) would become much easier to distinguish them. Method From 2010 to 2014, eleven pediatric patients with ML were enrolled (ALCL 3, T-LBL 3, B-LBL 1, Burkitt lymphoma 2, DLBCL 2). PET/CT findings before and after completion of chemotherapy or autologous/allogeneic hematopoietic stem cell transplantation (HSCT) were reviewed retrospectively. Results Diffuse thymic FDG uptake before chemotherapy was seen in 3 patients with T-LBL (mean SUV 7.9, range 5.5-12.5). Seven cases showed diffuse uptake in thymus after completion of therapy with relatively low SUV (mean SUV 3.4, range 2.2-5.2). We eventually considered all of them as rebound thymic hyperplasia and they are alive. On the other hand, one patient with nodular uptake in thymus (SUV 6.4) accompanied with high sIL-2R was finally diagnosed lymphoma recurrence by biopsy. The timing appearing thymic uptake after chemotherapy alone and high-dose chemotherapy with autologous HSCT were 2.8 months (range 1-7 months) and 8.0 months (range 6-10 months), respectively. In contrast, two patients receiving allogeneic HSCT did not show thymic hyperplasia anymore. Interestingly rebound thymic hyperplasia was persistently observed once it occurred and even in some cases the SUV increased slowly during follow period. The longest case in our cohort is 4.5 years at present time. Conclusion Diffuse thymic uptake with low SUV after chemotherapy could be rebound thymic hyperplasia unless relapse was suspected by any symptoms or abnormal laboratory findings. Rebound hyperplasia was frequently observed in pediatric patients, and once it occurs, it could last at least couple of years. More importantly, in case of nodular uptake, thymic recurrence should be strongly considered especially when accompanied with high SUV. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4302 Background and objective Conventionally, renal involvement in pediatric acute leukemia have been diagnosed by ultrasonography according to the definition of unilateral or bilateral nephromegaly based on the length of major or minor axis 〉2SD, diffuse abnormal findings, or mass legions. However, if renal involvements are determined just only by the length of major or minor axis〉2SD, overdiagnosis seems to be inevitable. Moreover, it might be difficult to detect renal mass legions by ultrasonography compared with other methods. To compensate for these shortcomings, in our hospital, we utilize contrast-enhanced CT to evaluate renal mass legions and to measure renal volume by utilizing 3D reconstruction models of CT imagings instead of measuring the length of major and minor axis of the kidney. Only when markedly decrease of renal volume before and after induction chemotherapy is observed, we define the nephromegaly at diagnosis as a true renal leukemic involvement. To assess whether our 3D-CT-based criteria for leukemic invasion to kidney could be superior to the conventional criteria, we retrospectively compared renal involvements in pediatric leukemia by conventional criteria with by 3D-CT-based criteria. Materials and Methods Between 2006 to 2012, 32 children were diagnosed with acute leukemia in our hospital. Enhanced CT scan was performed to the all patients before induction therapy, and a follow-up CT scan was performed after induction therapy or later stage on the patients who had abnormal CT findings at diagnosis. Mass legions, length of major and minor axis, and volume changes by utilizing enhanced CT and 3D reconstruction models calculated with the software SYNAPSE VINCENT (FUJIFILM MEDICAL), were evaluated. Results 21 out of 32 patients (65.6%) presented with renal involvement when conventional criteria was used (major or minor axis 〉2SD, or diffuse abnormal findings or mass legions). However, when applied to our 3D-CT- based criteria (mass legions or marked decrease of renal volume before and after induction therapy), only 6 patients out of 29 patients (20.7%) showed renal involvement (volume change alone 1, mass lesions without volume change 3, mass lesion with volume change 2 as shown in fig.1). The main reasons for the discrepancy between criteria for renal invasions were as follows. In the cases diagnosed with nephromegaly by satisfying major or minor axsis 〉2SD, only one case showed markedly decrease of renal volume before and after induction chemotherapy, and other cases did not show any renal volume changes and even in some cases, contrary to our expectation, renal volume increased as shown in fig.2. It suggested that the nephromegaly defined by only renal length include many false positive cases. Most importantly, renal mass legions could be detected only by enhanced CT, not by ultrasonography in some cases. Conclusion Our 3D-CT-based criteria for detection of leukemia invasion to kidney is much more reliable than the conventional criteria based on nephromegaly measured by major or minor axis. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4684 Mast cells have long been known as effector cells in the various IgE-mediated allergic responses. However, recent studies demonstrated that mast cells play various roles in immune reactions in coordination with other immune cells. That is, mast cells exert pro-inflammatory or anti-inflammatory effects depending on the situation. In addition, mast cells have association with tumor development. In allogeneic hematopoietic stem cell transplantation (HSCT), only a few have reported that the numbers of mast cells were correlated with the severity of acute GVHD in the skin. However, exact role of mast cells in GVHD remains unclear. With the purpose of potential application of mast cells in a clinical HSCT for GVHD, mixed lymphocyte reaction (MLR) was performed to clarify whether mast cells impaired the alloreaction or not. To generate bone marrow derived cultured mast cells (BMCMCs), BM cells from mice were incubated in complete RPMI containing IL-3 for 6 weeks. As shown in the figure, we showed that MLR was strongly inhibited when BMCMCs from the stimulator strain were added to the coculture (Stimulator (S): DCs obtained from C57BL/6, Responder (R): splenocytes from Balb/c, BMCMCs from C57BL/6). Next, when BMCMCs from the responder strain were added to the coculture, MLR was also suppressed (S: DCs obtained from C57BL/6, R: splenocytes from Balb/c, BMCMCs from Balb/c). In conclusion, mast cells suppressed lymphocyte proliferation induced by allo-DCs in an MHC-independent manner. Just like mesenchymal stem cells, cell therapy utilizing cultured mast cells may reduce GVHD severity. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1159 Hepatitis-associated aplastic anemia (HAA) is a variant of acquired aplastic anemia (AA) in which an episode of hepatitis precedes AA, namely hepatitis/aplastic anemia syndrome (HAAS). In AA, cytotoxic T-lymphocytes (CTL) play a central role in bone marrow (BM) destruction, and oligoclonal expansion of CTL correlates with disease activity. Immune mediated mechanisms have also been postulated in the pathogenesis of fulminant hepatitis (FH). Therefore we hypothesized that common CTLs recognizing similar target antigens against both liver and BM cells exist during the early period of FH, and subsequently selective proliferation of clones that are highly tropic to BM cause HAA at later stages. To this end, T cell phenotypes infiltrating liver, BM and peripheral blood (PB) samples obtained from 1-year-old boy with HAAS over time were determined by complementarity-determing region 3 (CDR3) size distribution patterns by polymerase chain reaction. Subsequently, CDR3 nucleotide sequences of predominant products were determined. Extremely skewed pattern was observed in Vb23 in BM and PB specimens at the onset of HAA (3 months after FH). A Vb23 clone with identical CDR3 nucleotide sequence predominated (clone 1) in BM and PB (34% and 60% within Vb23, respectively). This clone constituted a minor, but definitive clone in liver at the onset of FH (16% within Vb23). In contrast, initially predominant clones in liver (clone 3; 47% within Vb23) contracted to a minor subpopulation in BM and PB when the patient developed HAA (17% and 10% within Vb23, respectively). This patient had a very good response to immunosuppressive therapy (IST) using antithymocyte globulin, cyclosporine, and granulocyte-colony stimulating factor. Two months after starting IST, the pattern of CDR3 size distribution was just recovering to normal and initially predominant clones in both liver and BM almost disappeared in Vb23 in PB. However, extremely skewed pattern of CDR3 region was still observed and abnormal clone 1 had still occupied 71% within Vb23 in BM. As of 8 months after starting IST, CDR3 size distribution recovered to normal patterns (Gaussian distribution) and CTL clones against both liver and BM almost disappeared in both BM and PB. These suggest that it would take a relatively long time to recover immunological parameters completely even in IST good responders. In conclusion, our data is the first report to suggest that oligoclonal CTL clones simultaneously directed against liver and BM are involved in the pathogenesis of HAAS. Disclosures: No relevant conflicts of interest to declare.