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Graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a significant cause of morbidity and mortality. Regulatory T cells (Tregs) are critical mediators of immune tolerance after allo-HSCT. Clinical studies have indicated that programmed cell death 1 (PD-1) blockade before allo-HSCT involves a risk of severe GVHD. However, the mechanisms underlying GVHD induction resulting from PD-1 blockade remain unclear. We investigated the impact of PD-1 expression of donor T cells on T-cell reconstitution and GVHD using murine models. We first demonstrated that inhibition of PD-1 signaling induced aggressive expansion of CD4+ conventional T cells; however, Tregs could not maintain expansion because of high susceptibility to apoptosis, resulting in discordant immune recovery and subsequent development of severe GVHD. We then evaluated the impact of posttransplantation cyclophosphamide (PTCy) on abnormal T-cell reconstitution after PD-1 blockade. PTCy efficiently ameliorated GVHD after transplantation from a PD-1−/− donor and extended overall survival by safely regulating the proliferation and apoptosis of T-cell subsets. Notably, in the first 2 weeks after administration of PTCy, Tregs regained their ability to continuously proliferate, resulting in well-balanced reconstitution of donor T-cell subsets. In conclusion, the influence of PD-1 blockade differed within T-cell subsets and caused unbalanced reconstitution of T-cell subsets, resulting in severe GVHD. PTCy successfully restored T-cell homeostasis and ameliorated GVHD induced by PD-1−/− donor T cells. These findings may help explain the pathophysiology behind the observation that PTCy may mitigate the incidence and impact of GVHD associated with prior exposure to PD-1 blockade.

Graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a significant cause of morbidity and mortality. Regulatory T cells (Tregs) are critical mediators of immune tolerance after allo-HSCT. Clinical studies have indicated that programmed cell death 1 (PD-1) blockade before allo-HSCT involves a risk of severe GVHD. However, the mechanisms underlying GVHD induction resulting from PD-1 blockade remain unclear. We investigated the impact of PD-1 expression of donor T cells on T-cell reconstitution and GVHD using murine models. We first demonstrated that inhibition of PD-1 signaling induced aggressive expansion of CD4+conventional T cells; however, Tregs could not maintain expansion because of high susceptibility to apoptosis, resulting in discordant immune recovery and subsequent development of severe GVHD. We then evaluated the impact of posttransplantation cyclophosphamide (PTCy) on abnormal T-cell reconstitution after PD-1 blockade. PTCy efficiently ameliorated GVHD after transplantation from a PD-1−/−donor and extended overall survival by safely regulating the proliferation and apoptosis of T-cell subsets. Notably, in the first 2 weeks after administration of PTCy, Tregs regained their ability to continuously proliferate, resulting in well-balanced reconstitution of donor T-cell subsets. In conclusion, the influence of PD-1 blockade differed within T-cell subsets and caused unbalanced reconstitution of T-cell subsets, resulting in severe GVHD. PTCy successfully restored T-cell homeostasis and ameliorated GVHD induced by PD-1−/−donor T cells. These findings may help explain the pathophysiology behind the observation that PTCy may mitigate the incidence and impact of GVHD associated with prior exposure to PD-1 blockade.

In this paper, we propose a finite-time disturbance observer-based robust control method for output tracking of the Inteco three-tank system in the presence of mismatched uncertainties. The controller is designed in a backstepping manner. At each step of the virtual controller design, a robust feedback controller with some effective nonlinear damping terms is designed so that the system states remain in the feasible domain. The virtual controller is enhanced by a finite-time disturbance observer, and the dynamic surface control technique is adopted for the virtual controller to avoid the shortcoming of “explosion of terms”. Theoretical analysis is performed and simulation studies on the Inteco three-tank system model are included.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Allogeneic HSCT has a curative potential for patients with hematological malignancies. However, graft-versus-host disease (GVHD) remains to be a significant cause of morbidity and mortality after HSCT. Regulatory T cells (Tregs) are critical mediator for immune tolerance after HSCT and we recently reported that PD-1 plays an essential role for Treg survival (Asano et al, Blood 2017). Clinical studies suggested that PD-1 blockade prior to HSCT could be a risk of increasing severe GVHD. However, the mechanisms about GVHD induced by PD-1 blockade have largely unclear and there remains a paucity of data on appropriate GVHD prophylaxis for patients who undergo HSCT after PD-1 blockade. To address these issues, we investigated the impact of PD-1 expression on donor T cells on immune reconstitution with murine BMT models. First, lethally irradiated B6D2F1 mice were transplanted with 10 million of C57BL/6-background PD-1+/+or PD-1-/-spleen cells with 5 million of bone marrow cells from normal C57BL/6, and GVHD scores and overall survival was monitored. Recipients receiving PD-1-/-graft developed severe GVHD resulting in a significant shorter survival than recipients receiving PD-1-/-graft (P<0.0001). We analyzed lymphocytes in spleen and thymus on day3, 7, and 14. We found that CD8 T cells in PD-1-/-group showed markedly higher Ki67 expression and CFSE-dilution until day3. Interestingly, PD-1-/-Tregs increased aggressively at day3 but it could not maintain until day14, while PD-1-/-CD8 T cells and conventional CD4 T cells (CD4 Tcons) continued to increase until day+14, resulting in the significant higher CD8/Treg ratio in PD-1-/-group (P<0.05, vs PD-1+/+group). PD-1-/-Tregs showed significantly higher expression of Annexin V on day+7 and thymus CD4- and CD8- double-positive (DP) cells were in the extremely low levels in PD-1-/-group on day+14 (P<0.05, vs PD-1+/+group). Thymic analysis showed that donor PD-1-/-graft-derived CD8 T cells infiltrated thymus in PD-1-/-group, suggesting reconstruction of thymic function was critically disturbed by severe GVHD. These data suggest that loss of PD-1 signaling resulted in unbalanced reconstitution of donor-derived T cell subsets as a consequence of continuous CTL expansion and increased Treg apoptosis. Next, to evaluate the impact of post-transplant cyclophosphamide (PTCy) on the abnormal reconstitution after PD-1 blockade, we administered 50mg/kg of Cy or control vehicle on day3. PTCy efficiently ameliorated GVHD in PD-1-/-group and extended overall survival by safely regulating the proliferation and apoptosis of T cell subsets. Of note, after PTCy, Tregs regained the ability of continuous proliferation in the first 2 weeks, resulting in well-balanced reconstitution of donor-derived T cell subsets. Thymic DP cells on day 14 was markedly increased in PD-1-/-group with PTCy intervention as compared to without PTCy, suggesting PTCy could rescue thymus from PD-1 blockade-related severe GVHD. Finally, to evaluate GVL activity, we performed BMT with co-infusion of P815L tumor cells on day0 and we confirmed that PTCy treatment for PD-1-/-recipients reduced the severity of GVHD with maintaining sufficient GVL effect. In summary, our data suggested three insights about the impact of PD-1 signaling on immune reconstitution. First, PD-1 inhibition influenced graft-derived T cells very differently within T cell subsets. PD-1-/-Tregs increased transiently but it was counterbalanced by accelerated apoptosis, while PD-1-/-CD4+Tcons and CD8 T cells continued the drastic expansion. Second, we found that PD-1-/-donor T cells developed severe GVHD in thymus. Few reports have concentrated on the impact of donor graft PD-1 expression to thymus after BMT and acute GVHD in thymus could lead late central immune disturbance. Third, PTCy successfully ameliorated GVHD induced by PD-1-/-donor T cells preserving GVL effect. Cell proliferation study implied that PD-1-/-graft-derived CD8 T cells might be more susceptible for PTCy because of the high-rate proliferation. In conclusion, PD-1-/-graft cause lethal thymic GVHD and PTCy successfully ameliorated it. The influence of PD-1 inhibition was different within T cell subtypes. PTCy might be appropriate GVHD prophylaxis strategy for patients who had prior usage of PD-1 blockade.

No relevant conflicts of interest to declare.

Adult T-cell leukemia/lymphoma (ATLL) is highly aggressive CD4+ T cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). Normal cellular counterpart of ATLL is considered to be CD4+Foxp3+ regulatory T cells (Tregs) and therefore ATLL cell and Treg cell show similar phenotypic characteristics including the expression of CD25, Foxp3 and CCR4. Treg is immunosuppressive cells which has a critical role to suppress GVHD after allogeneic HSCT. Humanized anti-CCR4 monoclonal body, Mogamulizumab (Mog), is used for treatment of ATLL. We recently reported that pretransplant use of Mog was significantly associated with an increased risk of GVHD-related mortality (Fuji et al, JCO 2016). Since Tregs also express CCR4, the negative impact of Mog on transplantation tolerance was assumed to be a result from poor Treg recovery under the presence of Mog. However, it has not been well studied the interactional association of the sequential Mog concentration, the recovery of Tregs and the transition of residual ATLL cells after allogeneic HSCT in patients with peri-transplant administration of Mog. To tackle this issue, we here studied the clinical and laboratory sequences of patients who received Mog administration to control chemo-refractory ATLL in the peri-transplant period, as comparing patients without Mog treatment. Peripheral blood samples were obtained before and at 2, 4, 6, 8, and 12 weeks after HSCT and thereafter every 3 months. Total 7 patients were studied; 3 patients who received Mog before SCT, 1 patient who received Mog after SCT to treat early relapse of ATLL and 2 patients who did not received Mog. Tregs were defined as CD4+CD7+CADM1-CD25+CD127- and ATLL cells were defined as CD4+CD7-CADM1+, and these were compared with CD4+ conventional T cells (Tcons) in the same individual samples. Gated Tregs and ATLL cells were examined the expression of Foxp3, Helios, CCR4, CD45RA, CD31, Ki-67, BCL2, PD-1 and PD-L1. Mog concentrations in plasma were measured by ELISA. Plasma levels of TARC/CCL17 and MDC/CCL22, endogenous ligands of CCR4, were also measured. Our data indicated that peak levels of Mog were basically dependent of total administrated dose and residual ATLL cells were promptly reduced in blood. After the final dose of Mog, Mog levels were gradually declined (average of estimated half-life was 11.2 days). Shorter clearance rate was observed in a patient with high tumor burden (half-life 5.2 days) and in a patient received plasma exchange (half-life 8.4 days). All patients with Mog treatment failed the early expansion of Treg as compared with patients without Mog. Especially, CCR4-expressing effector-phenotype Treg was completely depleted from blood just after Mog treatment and 3 of 4 Mog-treated patients developed grade 2-4 GVHD. Of note, the delayed Treg recovery was prolonged over 12 months even after Mog was under detectable levels. After Mog decreased, concentrations of endogenous TARC and MDC were subsequently elevated in return. Expression of CCR4 on Treg was thereafter increased slowly but CD45RA+CD31+ recent-thymic emigrant population of Treg remained in the quite low levels over a year and the total Treg number was critically suppressed, suggesting that Mog treatment disturbed Treg homeostasis for a long period after HSCT. Taken together, our data demonstrated that both ATLL cells and Tregs are very susceptible to Mog treatment. Mog treatment not only exerts direct cytotoxicity on Treg but also appears to alter Treg homeostasis including the differentiation balance and distribution, at least in part, by the sequential enhancement of exogenous and endogenous ligand of CCR4. Our data might provide important information to develop safe and efficient HSCT for CCR4-positive ATLL and PTCL.

Posttransplant cyclophosphamide (PTCy) is an effective prophylaxis for both acute and chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). We recently studied the immune reconstitution dynamics of each lymphocyte subset after PTCy-based transplant using murine haploidentical BMT model and reported that PTCy strongly promoted Treg-dominant T-cell reconstitution and stem cell-derived mature B-cell generation with broad BCR-diversity. We also found that the early reconstitution of Treg could contribute to promote naïve B cell emergence from bone marrow, indicating the T and B cell recovery might be mutually coordinated after PTCy-based transplant (Iwamoto et al, ASH2017). However, the detailed process of immune reconstitution in patients after haploidentical HSCT with PTCy has not been well studied. To address this issue, we here investigated the early dynamics of donor-lymphocyte subset chimerisms in patient after clinical PTCy-based haploidentical HSCT with comparing those in patients after low-dose ATG-based haploidentical HSCT and patients after cord blood transplantation. Laboratory studies were undertaken in 13 adult patients who received HLA-mismatched allogeneic graft; unrelated cord blood (n=5), and haploidentical related peripheral blood after ATG-based conditioning (n=5) and haploidentical related peripheral blood after PTCy-based conditioning (n=5). Blood samples were obtained before and at 1, 2, 4, 6 and 8 weeks after HSCT. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by density gradient centrifugation and cryopreserved before being analyzed. After thawing, to analyze the subset-specific chimerism, PBMCs were stained with anti-HLA monoclonal antibodies and other subset-specific antibodies as follows: Pacific Blue conjugated anti-CD4, eFluor450 conjugated anti-CD3, PE-Cy7 conjugated anti-CD25, anti-CD14, APC conjugated anti-CD127, anti-CD56, and APC-eFluor780 conjugated anti-CD8a, anti-CD19. Gated lymphotes (CD4+Tcons, CD4+Tregs, CD8+T cells, B cells, NK cells, Monocytes) were analyzed their chimerism by flowcytometry. To examine the detailed phenotype of B cells, the expression of CD27, CD24, CD38 and IgD were tested. Flowcytometry-based method enables us to analyze the lymphocyte subset chemerism in the very early phase after HSCT. At 2 weeks after HSCT, our analysis revealed that CD4+Tcons, CD4+Tregs and CD8+T cells had already achieved complete donor chimerisms (>95% in all subsets) in patients after ATG-based SCT and had been approaching complete donor chimerisms (85.8%, 75.4% and 87.2%, respectively) in patients after CBT. In contrast, percentage of donor chimerisms of CD4+Tcons, CD4+Tregs and CD8+T cells after PTCy-based haplo-SCT was 73.5%, 59.6% and 59.2%, respectively, and those remained to be in the lower levels than other 2 groups. However, at 4 weeks after HSCT, all examined patients achieved complete donor chimerism of T cells, NK cells and Monocytes (>90%). At 8 weeks after HSCT, the number of B cells in PTCy-based haplo-group was higher than in ATG-based haplo-group (3494 vs 1901/mm3). Of note, B cell population in PTCy-based haplo-group at 8 weeks contained the significantly higher percentage of CD24+CD27-IgD+CD38+ transitional/naïve subset and the significantly lower percentage of CD24+CD27+IgD-CD38neg/dim activated/switched-memory subset when compared to B cell population in ATG-based haplo-group (59.9% vs 10.2%, 2.6% vs 21.5%, P<0.02 respectively), suggesting PTCy treatment might be associated with the favorable B cell reconstitution with naïve-subset dominant composition. Moreover, in patients after PTCy-based haplo-group, the percentage of activated/switched-memory subsets in B cell population at 8 weeks was inversely correlated with percentage of Treg in CD4 T cells at 4 weeks (P<0.05, r2=0.77). Taken together, consistently with our murine study, the current data from clinical samples again suggest that PTCy-based immune-modulation lead to coordinated T and B cell recovery, especially promoting naïve-subset dominant B cell recovery with help of the early expansion of Treg, which might reduce the risk of subsequent chronic GVHD. These data provide the important information for understanding the immunological reconstitution after PTCy-based haploidentical HSCT.

Adult T-cell leukemia/lymphoma (ATLL) is highly aggressive CD4+ T cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). Normal cellular counterpart of ATL is considered to be CD4+ regulatory T cells (Treg) and therefore ATL cell and Treg cell show similar phenotypic characteristics including the expression of CD25, Foxp3 and CCR4. Treg is immunosuppressive cells which contribute the maintenance of peripheral tolerance. Previous studies suggested that murine Treg show the distinct characteristics in cell-migration after activation, however, the expression of migration-related markers after activation on human Treg has not been well studied. In this study, we here examined the expression pattern of surface and intracellular markers including activation/migration receptors on Tregs from healthy individuals and Tregs and ATLL cells from 7 patients before and after allogeneic hematopoietic stem cell transplantation (HSCT). Peripheral blood samples were obtained before and at 2, 4, 6, 8, and 12 weeks after HSCT and thereafter every 3 months. Tregs were defined as CD4+CD7+CADM1-CD25+CD127- and ATLL cells were defined as CD4+CD7-CADM1+, and these were compared with CD4+ conventional T cells (Tcons). Gated Tregs and ATLL cells were examined for expression of CD45RA, CD62L, CCR4, CCR7, Ki-67, BCL2, PD-1 and PD-L1. Plasma TARC/CCL17 and MDC/CCL22, endogenous ligands of CCR4, were also measured by ELISA. First, we analyzed the expression of chemokine receptors of Treg from healthy donors. Our data showed that CD45RA+ naïve-type Treg express high level of CCR7 and little expression of CCR4 while CD45RA- activated/memory-phenotype Treg express low level of CCR7 and high level of CCR4. To elucidate the alteration of chemokine receptor expressions just after cell-activation in each Treg subpopulation, we sorted out 4 different CD4+ T cell subsets (CD45RA+ Tcon, CD45RA- Tcon, CD45RA+ Treg and CD45RA- Treg) from healthy donors, cultured with anti-CD3/CD28 antibodies and then measured the proliferation and the migration markers by CFSE-based assay. Tcons promptly decreased CCR7 after cell-division, in contrast, Tregs increased the expression of CCR7, suggesting Treg appears to have distinct migration attribute in which Treg may migrate to lymph node from periphery after activation. Next, we examined Tregs and ATLL cells from patients who received HSCT. 3 patients received mogamulizumab (Mog), a CCR4 monoclonal antibody, before HSCT and showed significantly lower residual ATL at HSCT as compared to 5 patients without pre-HSCT use of Mog (0.3% vs 15.8% of CD4+, P<0.05). Patients with pre-SCT Mog failed early expansion of Treg (3.0% vs 14.6% of CD4+ at week4, P<0.05) and the delayed recovery was prolonged over 12 months. 3 patients developed relapse. PD-L1 on ATL cells was negative at HSCT in these patients but was strongly expressed at relapse. The expression of CD25 was low at the early stage of relapse, which was later upregulated. Interestingly, relapsed ATLLs involved CD45RA+CD62L- or CD45RA-CD62L- subset with high CCR7 and Ki-67 and the slightly decreased CCR4, those are very similar to normal Treg after activation. TARC and MDC were highly elevated just after the treatment but was the elevation was transient. Our data demonstrated that Tregs and ATLL cells showed the similar expression of migration markers on activation and it is clearly different from conventional T cells. ATLL cells at relapse after HSCT showed the decreased skin-oriented CCR4 and the increased lymph node-oriented CCR7, suggesting activated ATLL cells might escape from the immunological surveillance by GVL which is actively exerted in GVHD-target tissues including skin. Our data might provide important information to develop efficient HSCT for ATLL.

Posttransplant cyclophosphamide (PTCy) is an effective prophylaxis for both acute and chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Recent studies reported that PTCy has been associated with low incidence of viral infections or EBV-related posttransplantation lymphoproliferative disease (EB-LPD), suggesting PTCy-based immune modulation leads the favorable immune reconstitution after transplant. Recently, we studied the immune reconstitution dynamics of each lymphocyte subset after transplant using PTCy and reported that PTCy-based immune-modulation strongly promoted Treg-dominant T-cell reconstitution and stem cell-derived mature B-cell generation by using murine model of haploidentical HSCT with PTCy. TCR- and BCR- repertoire analysis demonstrated that PTCy resulted in the broader diversity of both T cell and B cell repertoire than non-PTCy controls. However, the mechanism of coordination of increased Tregs and B cells after PTCy-based HSCT has not well studied. To address this issue, we here investgated the impact and role of PTCy on Tregs and B cell recovery and the mutual influence between the two important subsets by using murine BMT model. Irradiated B6D2F1 mice were transplanted with 5x106spleen cells from the CD45.1 B6 mice together with 5x106TCD-BM from CD45.2 B6 donors. Cyclophosphamide 100mg/kg or control vehicle was administered at day 3 after transplant. Peripheral blood mononuclear cells (PBMCs) and splenic cells were sequentially obtained at day21. The chimeric balances among host-residual H-2kd+ cells, donor graft-derived cells and donor BM-derived cells in Tregs and B cells were monitored separately. To evaluate the homeostatic stability, proliferation marker Ki-67 and anti-apoptotic BCL-2 were also quantitatively examined respectively. To examine the detailed phenotype of Tcells, CD4, CD8, CD25, Foxp3, ICOS, CXCR5 were tested. Also, to examine the detailed phenotype of B cells, the expression of B220, CD21, CD23, CD24 were tested. At day21 after HSCT, the number of Treg cell in spleen was significantly higher on PTCy-treated recipients than that of non-PTCy control (P < 0.05). We also checked the emergence of donor-derived follicular helper T cells (Tfh) in lymph node because recent studies have shown that Tfh could contribute B cell differentiation after HSCT. In our analysis at day21 in this PTCy model, Tfh cells were still few in both PTCy group and non-PTCy group, suggesting the early T cell recovery after PTCy did not promote B cell differentiation into pathological plasmablast. The number of B cell was markedly higher in PTCy-treated recipients than that of non-PTCy control (P < 0.01). The increased B cell population was mainly transitional-1 phenotype suggesting these B cells were recent emigrants from bone marrow. In consistent with low recovery of Tfh cells, plasmablast-like phenotype of B cells were not observed. Next, to evaluate the impact of graft-derived Treg recovery on B cell reconstitution, we depleted Treg from spleen cells of B6 donors by using CD25+ magnetic beads. After the depletion, residual Treg cell in the graft was under 1% of total CD4 T cells. At day21, splenic Treg cells is under 3% of total CD4 T cells in Treg-depleted group, that is much lower than Treg in Treg-repleted group. Interestingly, the number of B cells at day21 in PTCy-treated recipients was equivalent to that of non-PTCy recipients when we depleted Treg from donor graft, suggesting that well-balaced B cell recovery promoted by PTCy was completely abolished by Treg-depletion. Taken together, our data suggested that PTCy could induce the expansion of Treg recovery and naïve B cell recovery without urging early emergence of Tfh cells in lymph node, resulting in well-diversed T and B cell reconstitution. Treg appears to contribute to promote naïve B cell emergence from bone marrow, indicating the T and B cell recovery might be mutually influenced. Moreover, slow recovery of Tfh cells in lymph node could be advantage for preventing B cell from forced differentiation into plasmablast causing GVHD. These data provide the important information for understanding the immunological reconstitution after PTCy-based haploidentical HSCT.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the curative treatment of many hematological malignancies, but the relapse is one of the major causes of treatment failure and mortality. Previous clinical studies have shown that low-dose IL-2 was well tolerated and could lower the relapse rate in human T cell depleted allogeneic BMT. On the other hand, we also demonstrated that low-dose IL-2 could preferentially enhance regulatory T cell (Treg) and improve the symptoms of chronic GVHD (NEJM 2011,Sci Trans Med 2013). In murine bone marrow transplantation (BMT) model showed that Treg can protect from lethal GVHD while preserving graft-versus-leukemia (GVL) effect. Especially, CD62L+ Tregs has a higher capacity in suppressing GVHD than their CD62L- counterpart. Our recent study reported that exogenous low-dose IL-2 induced the expression of PD-1 on Treg preferentially and it was most evident in CD44+CD62L+central-memory Treg (Blood 2016). However, the impact of CD62L+ Treg enhanced by low-dose IL-2 on GVHD and GVL has not been well investigated. In this study, we studied the effect of exogenous IL-2 on the lymphocyte phenotypic profiles including naïve- versus memory-type balance and also examined donor-derived immunity, especially GVHD and GVL, by using leukemia-bearing BMT model. Lethally irradiated B6D2F1 recipients were transplanted spleen cells and BM cells from B6 donors and 5000 IU Teceleukin (recombinant IL-2) was injected once a day for 14 days. The number of CD4+CD25+Foxp3+ Treg, CD4+CD25-Foxp3- conventional T cell (Tcon) and CD8+ T cell was compared between IL2- and vehicle-treated groups. The expressions of CD44, CD62L, CCR4, CCR7, Ki-67, PD-1, CTLA-4, LAG3, GITR, ICOS and TIM-3 in each subset were also examined. Lymphocytes profiling analysis showed that IL-2 treatment in the first 7 days resulted in the dominant expansion of effector T cells without Treg increase (Treg 0.10 vs 0.16,p=0.35, Tcon 2.83 vs 4.20, p=0.01, CD8 T cell 5.52 vs 10.4, p=0.01, million). On the contrary, the extended IL-2 treatment in the next 7 days increased CD62L+ Treg (21.5 vs 48.2 % of total Treg, p<0.001, 0.02 vs 0.07 million, p=0.006) without increasing effector T cells. PD-1 on Treg was very high in BMT-recipients even without receiving IL-2, so further enhancement of PD-1 expression by IL-2 was not observed. However, the expression of other inhibitory molecules including CTLA-4 (MFI, 6.61 vs 9.92, p=<0.001), LAG-3 (1.73 vs 2.17, p=0.04) and ICOS (2.41 vs 3.84, P<0.001) was enhanced by the IL-2 treatment. To elucidate the mechanisms of the increase in CD62L+ Treg, the in vivo violet dye dilution assay was performed. We first sorted out CD62L+ T cells by micro-beads from spleen cells, and then adaptively transferred into recipients. Five days treatment of low-dose IL-2 resulted in the enhancement of the division of CD62L+ Treg (% divided cells; 19.5 vs 29.8 % of Treg, p=0.005, 62.3 vs 108 in number, p=0.002). Interestingly, when CD62L- T cells were transferred, IL-2 treatment also induced the division of the cells and, of note, induced the phenotypic change of CD62L- into CD62L+ Treg (7.63 vs 19.8 % of Treg, p=0.009, 91.3 vs 361, p=0.01). These results suggested that IL-2 have the possibility to enhance the proliferative activity of Treg with the up-regulation of CD62L expression. Lastly, we transplanted lucipherase+P815 leukemia cells into this BMT model and evaluated the clinical impact of low-dose IL-2 on GVL by using in vivo bioluminescence system. To assesse the GVL and GVHD independently, we used two different tumor burden models. In the high tumor burden model, IL-2 injection decreased bioluminescence intensity (BLI) of P815 (BLI at week3, 8.3x10E7 vs 8.1x10E6 photon/second, p<0.001) and significantly extends overall survival. On the other hand, in the low tumor burden model, both treatment groups did not die from leukemia but vehicle-treated control group showed a significant higher clinical GVHD score and died from GVHD. These data demonstrated the clinical benefits on anti-tumor effect of IL-2, which could enhance the GVL activity without the detrimental effect on GVHD. In conclusion, our data indicate multiple effects of IL-2 on post-transplant immunity can coordinate to intensify GVL separately from GVHD on the mechanism of sequential enhancement of effector T cell and CD62L+ Treg in the different phases. These data provide the important information for developing Treg-targeted therapy.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the curative treatment for many hematological malignancies. Clinical studies have shown that HSCT from haplo-identical donors with posttransplant cyclophosphamide (PTCy) can widely provide alternative donor sources and is an effective prophylaxis for both acute and chronic graft-versus-host disease (GVHD). However, on the other hands, it is concerned that the immune suppressive effect by PTCy also might diminish graft-versus-leukemia (GVL) effect and increase the relapse rate in patients with high tumor burden at transplant. Indeed, recent clinical studies suggested that a certain extent of dose-reduction of PTCy could sufficiently prevent GVHD, but whether the dose-reduction can lead to enhance GVL effect has not been clarified. In this study, we investigated the effects of dose-reduction of PTCy on GVHD and GVL by using murine haploidentical BMT model. Also, we explored the immune-modulating impacts of liposomal alpha-galactosylceramide (liposomal a-galcer; RGI2001) on reduced PTCy. Liposomal a-galcer is a synthetic glycolipid, a ligand stimulating invariant natural killer T (NKT) cell in CD1d restricted manner, which is shown to prevent GVHD via expansion of regulatory T cell (Treg) in murine experiments. First, we tested the effects of dose-reduction of PTCy on GVL effect, we transplanted from C57BL/6J donor to B6D2F1 recipients bearing with luciferase-expressing P815 leukemia cells. The impact of PTCy dose-reduction on GVL was evaluated by using in vivo bioluminescence system. Lethally irradiated B6D2F1 mice were transplanted with 5x106T cell depleted (TCD) BM, 10x106whole splenic cells from C57BL/6J donor mice and 5x105luciferase-expressing P815 leukemia cells. PTCy or control vehicle was administered at day3 after BMT. As for PTCy, 50mg/kg of cyclophosphamide (Cy) has already shown as standard dose to sufficiently suppress GVHD, thus we tested effect of 25mg/kg of Cy comparing with 50mg/kg of Cy. In vivo imaging system (IVIS) was followed weekly since day10 after BMT to evaluate tumor burden. A trend toward lower levels of radiance was measured by IVIS in reduced-dose group than full-dose group through the experimental period, suggesting reduced-dose of Cy contribute to leave alloagressive T cell activity and result in the enhancement of GVL effect. However, the benefit of GVL enhancement in reduced PTCy group was counterbalanced by the deterioration of GVHD when we used higher dose of total body irradiation (TBI) for the aggressive GVHD-prone experimental setting. To overcome the negative impact of PTCy-reduction on GVHD, we next tested the immune-modulating effect of liposomal a-galcer on this system. Liposomal a-galcer 1mg/kg or control vehicle was administered at day3, 5, 7, after BMT. Our data indicated that reduced PTCy plus liposomal a-galcer significantly improved acute GVHD in overall survival (vs control group, p < 0.01). In comparison with those treated with PTCy plus liposomal a-galcer, the others kept on losing weight and worsening GVHD score to the end beyond the third week without recovery. Importantly, liposomal a-galcer did not influence the effect of GVL when we evaluated tumor-bearing recipents with IVIS, suggesting that add-on of liposomal a-galcer improved GVHD with remaining intensified GVL effect which was gained by dose-reduction of PTCy.　Flowcytometric analysis of splenic cells at day 14 showed that %Treg of CD4+ Tcell was significantly higher in reduced PTCy plus liposomal a-galcer group than control group (5.30 vs 2.75, p < 0.05) and reduced PTCy alone group (5.30 vs 2.57, p < 0.05). Additionally, in PTCy plus liposomal a-galcer group, both Treg/CD4+ conventional Tcell (Tcon) ratio and Treg/CD8+ Tcell ratio were significantly higher than control group (p < 0.05) and reduced PTCy alone group (p < 0.05), respectively. Analysis of MLN cells also showed the selective increase of Treg in PTCy plus liposomal a-galcer group more than in other groups. Analysis of splenic cells and MLN cells at day28 showed similar trends with at day14. Taken together, these data demonstarated that liposomal a-galcer could efficiently compensate the immune-suppressive effect after reduced PTCy by preferentially promoting Treg recovery with remaining GVL. Our data might provide the important information for developing new strategy of haploidentical HSCT with PTCy for patient with high-tumor burden.

No relevant conflicts of interest to declare.

Introduction: Hematological complete remission (CR) is the evident prognostic factor of allogeneic hematopoietic stem cell transplantation (HSCT) in patients with Acute Myeloid Leukemia (AML). CR with incomplete blood cell count recovery (CRi) after induction therapy was identified as independent prognostic factor for inferior long-term outcomes in patients with AML achieving remission (Chen et al, JCO 2015). There is a paucity of data regarding the impact of response (CR vs. CRi) prior to allogeneic HSCT. Here we examined whether CRi provide prognostic information on the transplant outcomes.

Maeda: Mundipharma KK: Research Funding.

Introduction:The creatinine clearance rate (Ccr) is a more accurate indicator of renal function than serum creatinine. There remains a paucity date of prognostic value of mild to severe renal impairment calculated by Ccr for patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). Herein, we evaluated the transplant outcome of patients with mild to severe renal dysfunction and compared to those without renal dysfunction.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Introduction: Allogeneic hematopoietic cell transplantation (HCT) has curative potential for a variety of hematologic malignancies. However, the success of HCT is heavily dependent on the disease and remission status. Armand et al. recently proposed a disease risk index (DRI) to assess the risk for allogeneic HCT based on the disease and remission status, and could be used to risk stratification on survival (Armand et al Blood 2012), but evaluation of its clinical significance is limited. HCT-comorbidity index (HCT-CI) was developed as a measure of pretransplant organ dysfunctions (Sorror et al Blood 2005), which has been shown to be associated with nonrelapse mortality (NRM). In order to clarify any association between pretransplant factors including DRI and HCT-CI, and respective overall survival (OS), NRM and relapse rate, we retrospectively reviewed the data of patients who underwent allogeneic HCT at our department.

Background: Previous studies have shown that the absolute lymphocyte count (ALC) in peripheral blood at diagnosis may be an independent prognostic factor of IPI for patients with diffuse large B-cell lymphoma (DLBCL). In the rituximab era, the U.S. National Comprehensive Cancer Network International Prognostic Index (NCCN-IPI) was developed to improve the risk stratification of DLBCL in comparison to the existing IPI. Therefore, the aim of this study was to clarify the impact of ALC at diagnosis on event free survival (EFS) and overall survival (OS) on analysis performed with factors included in NCCN-IPI.

Background:A consensus is yet to be reached on therole of interim fluorine-18 fluorodeoxyglucose (18F-FDG) position emission tomography-computed tomography (PET-CT) for prognosis in diffuse large B-cell lymphoma (DLBCL), but it has been recently reported that maximum standardized uptake value (SUV max) reduction between baseline and midtherapy may be a better predictor of overall survival (OS) and event free survival (EFS).

The prognosis of elderly patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) is poor, even in the patients who achieved good response after induction therapy. In order to undertake allogeneic stem cell transplantation (allo-SCT) with reduced toxicity and without total body irradiation (TBI), we conducted a combination regimen consisted of fludarabine, busulfan, and melphalan (Flu/Mel/Bu) for reduced intensity stem cell transplantation (RIST) conditioning.Among a total of 50 patients who underwent Flu/Mel/Bu conditioning between 2004 and 2012 in our institute, 32 patients with myeloid malignancies were retrospectively reviewed. Disease status was defined by WHO classification 2008. Therapy consisted of fludarabine 25 mg/m2 for five days (125mg/m2) and melphalan 50mg/m2 for two days (100mg/m2), both by intravenous infusion. Busulfan 2 mg/kg was administered orally for two days (4mg/kg) between 2004 and 2006, and intravenously at 1.6 mg/kg for two days (3.2mg/kg) between 2007 and 2012.Among the 32 eligible patients, 18 were female and 14 male. Seventeen patients were diagnosed with AML, 14 with MDS and one with chronic myelomonocytic leukemia (CMML). Median age was 59 years (32-66 years), and the median follow-up period was 1337 days (12-3043 days).Disease status of AML was complete remission (CR)1 (5), CR2 (10), CR3 (1) and CR4 (1), respectively, and all CR1 patients had poor risk factors. Disease status of MDS was RA (4), RARS (2), RCMD (1), RAEB-1 (5) and RAEB-2 (2), respectively. Three patients of RAEB conducted induction chemotherapy and two patients achieved CR. Donor sources consisted of 22 of unrelated bone marrow (URBM), 5 of related bone marrow (RBM), 3 related peripheral blood (RPB), and 2 of unrelated cord blood (URCB), respectively. Graft-versus-host disease (GVHD) prophylaxis consisted of tacrolimus plus short term methotrexate (TAC+sMTX) (25) and cyclosporin plus methotrexate (CsA+sMTX) (7). The cumulative incidence of grade II-IV acute GVHD was 31.7% and chronic GVHD was 85.9%. Five-year non-relapse mortality (NRM) was 15.9% and the 5-year relapse rate was 18.8%. One-year overall survival (OS) was 81.2% (95%CI 62.9-91.1), 5-year OS was 74.6% (95%CI 55.5-86.4), one-year progression free survival (PFS) was 78.1% (95%CI 59.5-88.9), 5-year PFS was 65.4% (95%CI 44.2-80.2). Fourteen patients were older than 60 years, and both 5-year OS and PFS of this group were 85.7% (95%CI 53.9-96.2). For AML, one-year OS was 82.4% (95%CI 54.7-93.9), 5-year OS 70.1% (95%CI 42.3-86.3), one-year PFS was 76.5% (95%CI 48.8-90.4) and 5-year PFS was 61.8% (95%CI 32.9-81.2). Five-year NRM was 11.8% and the 5-year relapse rate was 26.5%. For MDS, both one-year OS and 5-year OS were 78.6% (95%CI 47.2-92.5), and both one-year PFS and 5-year PFS were 69.8% (95%CI 37.8-87.6). Five-year NRM was 21.4% and the 5-year relapse rate was 8.7%.On the other hand, we conducted 32 allo-SCT for myeloid malignancies (22 of AML in CR and 10 of MDS) with conventional conditioning regimens of cyclophosphamide (Cy) and TBI or Bu and Cy between 2004 and 2012. Median age was 36.5 years (20-54 years), and the median follow-up period was 1191 days (38-3366 days).Disease status of AML was CR1 (14), and CR2 (8), respectively. Disease status of MDS was RA (3), RARS (2), RCMD (1), RAEB-1 (4), RAEB-2 (1), and MDS-U (1), respectively. Four patients of RAEB conducted induction chemotherapy and achieved CR. Donor sources were URBM (15), RBM (11), CB (5) and RPB (1), respectively. GVHD prophylaxis consisted of TAC+sMTX (17) and CsA+sMTX (15).The outcomes of Flu/Mel/Bu were not statistically inferior to those of conventional regimens with one-year OS of (81.2% vs. 87.1%, p=0.564) and 5-year OS of (74.6% vs. 78.0%, p=0.564), and one-year PFS of (78.1% vs. 83.9%, p=0.183) and 5-year PFS of (65.4% vs. 80.4%, p=0.183).Flu/Mel/Bu was tolerable, and produced good outcomes and may be a candidate for curative conditioning regimen of RIST, especially for patients with myeloid malignancies in controlled status.No relevant conflicts of interest to declare.

The prognosis of elderly patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) is poor, even in the patients who achieved good response after induction therapy. In order to undertake allogeneic stem cell transplantation (allo-SCT) with reduced toxicity and without total body irradiation (TBI), we conducted a combination regimen consisted of fludarabine, busulfan, and melphalan (Flu/Mel/Bu) for reduced intensity stem cell transplantation (RIST) conditioning.

The Wilms' tumor gene (WT1), originally discovered as a tumor suppressor has been proven to have an oncogenic role in leukemia and several other cancers. WT1 mRNA expression levels in peripheral blood (PBWT1) has been reported as a useful marker for the risk evaluation of myelodysplastic syndrome (MDS). In the era of hypomethylating agents, the significance of PBWT1 on MDS prognosis is still unknown. This study aimed to clarify the impact of pre-treatment PBWT1 levels on overall response (OR) and overall survival (OS) in MDS patients treated with azacitidine (AZA).We retrospectively analyzed all patients from March 2011 to March 2013 with World Health Organization 2008 defined MDS, CMML or AML with 20–30% bone marrow blasts who received AZA treatment in our department for at least one cycle (37.5–75.0 mg/m2/day during 7 days, every 28 days). Patients' peripheral blood specimens were collected before AZA initiation, mRNA was extracted from leukocytes using the RNeasy Mini-Kit (Qiagen, Valencia, CA), and the amount containing WT1 mRNA was measured using a WT1 mRNA Assay Kit (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan). Hematologic response was evaluated according to International Working Group 2006. OR was defined as a best overall response of complete remission (CR), partial remission, marrow CR, or hematologic improvement. Univariate analyses for OR were carried out using Fisher's exact test. Factors associated with at least borderline significance (p < 0.10) were subjected to a multivariate analysis, using logistic regression model. OS was estimated according to the Kaplan-Meier method. Multivariate analysis was performed with proportional hazard Cox model, including all variables with p < 0.10 in univariate analyses.Of 55 patients enrolled, pre-treatment PBWT1 levels were available in 41 patients and the median level was 790 copies/µg RNA (range, less than 50–310000). Baseline characteristics according to PBWT1 levels (≤ 790 [lower group] [n = 21] and > 790 [higher group] [n = 20]) are summarized in Table 1. Median number of AZA treatment cycles was 4 (range, 1–18). Four patients (2 in higher group, and 2 in lower group) received allogeneic stem cell transplantation (alloSCT) after AZA treatment. OR rates were significantly lower in PBWT1 higher group than lower group (30.0 vs 71.4%, p = 0.03). In univariate analysis, other significant risk factors or with borderline significance for OR were higher serum ferritin levels (> 1000 ng/ml) and RBC transfusion dependency ≥ 4 units/8 weeks. In multivariate analysis, higher PBWT1 levels independently predicted reduced likelihood of OR (odds ratio = 0.212, 95% CI 0.01-0.95, p = 0.02). OS was significantly inferior in PBWT1 higher group as shown in Figure 1. In univariate analysis, other significant factor was Revised International Prognostic Scoring System (IPSS-R) risk groups (high risk defined as IPSS-R high or higher, and low risk defined as IPSS-R intermediate or lower). In multivariate analysis, higher PBWT1 levels (hazard ratio [HR] = 9.75, 95% CI 1.22-77.58, p = 0.03) and IPSS-R high risk (HR=7.04, 95% CI 1.43-34.48, p = 0.02) were independent predictors for OS.Our results suggest that PBWT1 can predict both response and survival of MDS patients treated with AZA. Although salvage therapy including alloSCT can affect the survival, poor survival might result from inferior response rates in PBWT1 high patients. In MDS with high PBWT1, restoration of epigenetically silenced tumor suppressor genes with AZA might not induce apoptosis. We propose that alternative therapeutic strategies should be sought in MDS patients with high PBWT1 levels.No relevant conflicts of interest to declare.

The Wilms' tumor gene (WT1), originally discovered as a tumor suppressor has been proven to have an oncogenic role in leukemia and several other cancers. WT1 mRNA expression levels in peripheral blood (PBWT1) has been reported as a useful marker for the risk evaluation of myelodysplastic syndrome (MDS). In the era of hypomethylating agents, the significance of PBWT1 on MDS prognosis is still unknown. This study aimed to clarify the impact of pre-treatment PBWT1 levels on overall response (OR) and overall survival (OS) in MDS patients treated with azacitidine (AZA).

Hemorrhagic cystitis (HC) is a major complication after allogeneic hematopoietic stem cell transplantation (SCT), and is life-threatening in severe cases. Early onset HC is usually due to toxic effects of conditioning regimen, while late onset HC is caused by viral or bacterial infection, and GVHD. In order to find out risk factors and to analyze prognosis of late onset HC, we retrospectively reviewed data in our institution from 1996 through 2009.We analyzed 232 cases having underwent allogeneic SCT (AML; 65, MDS; 42, ALL; 44, ML; 44, others; 37), excluding 22 patients who received second allogeneic transplantation. The median age at SCT was 47 years old (range, 13–72), and median follow-up of survivors was 1529 days (range, 99–4822) after SCT. One-hundred and eleven patients received from related donor, and 121 from unrelated. Conditioning was myeloablative in 139 cases, and reduced intensity (RIC) in 93. Myeloablative regimen was cyclophosphamide (CY) plus total body irradiation (TBI) in 79 cases, and busulfan (BU) plus CY in 54, while RIC regimen was fludarabine (Flu) plus melphalan (Mel) plus BU in 52, and Flu plus Mel plus TBI in 12. Disease status at SCT was controlled (partial or complete remission) in 164 cases, and progressive in 68. Prophylaxis for acute GVHD consisted of calcineurin inhibitor (cyclosporine; 106 cases, tacrolimus; 126 cases), short term methotrexate (158 cases), and corticosteroid (73 cases). Acute GVHD occurred in 121 cases, 92 of them were grade II-IV, and 74 were treated with corticosteroid. Cytomegalovirus (CMV) antigenemia was positive in 82 cases after engraftment. Definition of late onset HC was microscopic or macroscopic hematuria with urinary symptoms occurring after 7 days from SCT. Urine culture and virological analysis (polymerase chain reaction of CMV, adenovirus [ADV], and BK virus DNA) were carried out in all cases with HC. Treatment was mainly hydration and intravenous hemostat, including intravenous immunoglobulin (IVIg) and antiviral agents.HC was reported in 43 cases on a median of 36 days (range, 7–469) after SCT, and 31 of them (72.1%) recovered after a median of 21 days (range, 2–252). Cumulative incidence of HC after 1 year from SCT was 21.6% (95% confidence interval [CI] 18.3–24.7%). In univariate analysis, risk factors identified included age over 45 years old (p=0.04), progressive disease status at SCT (p=0.04), myeloablative conditioning (p=0.04), acute GVHD grade II-IV treated with corticosteroid (p=0.01), and positivity of CMV antigenemia after SCT (p<0.01). On multivariate analysis, older age (p<0.01, hazard ratio [HR] 2.63), myeloablative conditioning (p<0.01, HR 4.32), and positivity of CMV antigenemia (p<0.01, HR 3.61) remained independent predictors. Each HC case was graded according to National Cancer Institute Common Terminology Criteria for Adverse Events (grade 1; 12, grade 2; 19, grade 3; 12, grade 4 to 5; none), and we defined moderate as grade 1 or 2, and severe as grade 3 or more. In subgroup analysis of patients with HC, myeloablative conditioning (p=0.11, Odd's ratio [OR] 5.24) and positivity of ADV in urine (p=0.10, OR 3.17) tended to be associated with severe HC. Recovery rate from HC was inferior and median duration from onset to recovery was longer in severe HC (25.0%, 121 days) with significance (p<0.01) compared with moderate HC (90.3%, 17.7 days). Using of IVIg or antiviral agent (vidarabine, gancyclovir, or foscarnet) did not improve recovery rate in both groups. Overall survival (OS) at 1 year after day 35 landmark had no significance between cases with and without HC (59.7%, 56.7%, p=0.70), significantly poor in severe cases (16.7%, p<0.01) compared to cases with moderate HC and cases without HC. Treatment-related mortality (TRM) at 1 year after day 35 landmark was 25.0% with HC and 29.0% without HC (p=0.35), while 59.1% in patients with severe HC (p=0.01).Five significant risk factors of HC such as older age, progressive disease status, myeloablative conditioning, acute GVHD, and CMV antigenemia were found out in this study. OS and TRM of patients with all the grade of HC were equivalent to those without HC, while severe HC significantly elevated TRM and shortened OS. Severity of HC was related to myeloablative regimen and ADV in urine. Any successful attempts to prevent severe HC have not been established yet, and novel prophylactic and pre-emptive strategies are warranted.No relevant conflicts of interest to declare.

Abstract 2304