ALBERT

Description: Abstract 2451 Poster Board II-428 Hematopoietic cell transplantation (HCT) is a curative therapy for a variety of malignancies. HCT provides disease eradication through both the high-dose conditioning regimen and an allogeneic graft versus tumor effect (GVT), however graft-versus-host disease (GVHD) remains a major obstacle. In a murine aHCT model of bioluminescence imaging (BLI) we have previously demonstrated that acute GVHD can be separated to a GVHD initiation phase confined to secondary lymphoid organs and a subsequent GVHD effector phase in peripheral target tissues. It has been proposed that host conditioning may not only be crucial in the activation of alloreactive T cells but also determine acute GVHD organ manifestation in the effector phase. Here we wanted to investigate how the host conditioning regimen affects the host target tissues in terms of inflammatory cytokines and their role in donor T cell recruitment. We compared lethally irradiated (8Gy) vs. non-irradiated Balb/c wild type or Balb/c Rag-/-cGC-/- (H-2d) -DKO mice that received allogeneic luciferase+ FVB/N T cells (H-2q). Surprisingly, we did not observe marked differences in the donor T cell proliferation (BLI, CFSE), acquisition of activation markers (CD25, CD44, CD69) and homing receptors (a4b7, aEb7, P-selectin ligand, E-selecting ligand) in conditioned, non-conditioned Balb/c Rag-/-cGC-/-. Despite the upregulation of these homing receptors on donor T cells, infiltration of target tissues (intestinal tract, liver and skin) was significantly accelerated in conditioned and delayed in non-conditioned hosts. As T cell recruitment may have occurred as a result of alterations of the milieu inflammatory cytokines in GVHD target tissues, we compared the cytokine profile in conditioned vs. non-conditioned recipients. At days 3 and 6 after transplantation tissues were harvested and cytokines from the target tissues; liver, large bowel, small bowel, peripheral blood and a non target tissue: kidney were analyzed for a TH1/TH2/Th17a cytokines. At day 3 high levels of INF-γ and TNF were detected in the Balb/c WT conditioned host compared to the non-conditioned host in all target tissues (SB, LB, and liver) and most markedly in peripheral blood and the large bowel. More importantly the Balb/c Rag-/-cGC-/- conditioned host displayed about 5 times higher levels of both inflammatory cytokines compared to the non conditioned DKO hosts and to the Balb/c WT. Similar results with a lesser levels were observed both for IL-2 and IL17a. By day 6 similar results are seen but with a much reduced expression of the cytokines, indicating that the cytokine storm peak was maybe at day 3. In summary host conditioning is not a requirement for alloreactive T cell activation rather induced inflammatory cytokines such as TNF and INF-γ are the determinant factors for effector T cell recruitment to GVHD target tissues. JB and AB contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

Description: In acute graft-versus-host disease (aGVHD), donor T cells attack the recipient's gastrointestinal tract, liver, and skin. We hypothesized that blocking access to distinct lymphoid priming sites may alter the specific organ tropism and prevent aGVHD development. In support of this initial hypothesis, we found that different secondary lymphoid organs (SLOs) imprint distinct homing receptor phenotypes on evolving alloreactive effector T cells in vivo. Yet preventing T-cell entry to specific SLOs through blocking monoclonal antibodies, or SLO ablation, did not alter aGVHD pathophysiology. Moreover, transfer of alloreactive effector T cells into conditioned secondary recipients targeted the intestines and liver, irrespective of their initial priming site. Thus, we demonstrate redundancy of SLOs at different anatomical sites in aGVHD initiation. Only prevention of T-cell entry to all SLOs could completely abrogate the onset of aGVHD.

Description: Graft-versus-host disease (GVHD) is a major obstacle in allogeneic hematopoietic cell transplantation. Given the dynamic changes in immune cell subsets and tissue organization, which occur in GVHD, localization and timing of critical immunological events in vivo may reveal basic pathogenic mechanisms. To this end, we transplanted luciferase-labeled allogeneic splenocytes and monitored tissue distribution by in vivo bioluminescence imaging. High-resolution analyses showed initial proliferation of donor CD4+ T cells followed by CD8+ T cells in secondary lymphoid organs with subsequent homing to the intestines, liver, and skin. Transplantation of purified naive T cells caused GVHD that was initiated in secondary lymphoid organs followed by target organ manifestation in gut, liver, and skin. In contrast, transplanted CD4+ effector memory T (TEM) cells did not proliferate in secondary lymphoid organs in vivo and despite their in vitro alloreactivity in mixed leukocyte reaction (MLR) assays did not cause acute GVHD. These findings underline the potential of T-cell subsets with defined trafficking patterns for immune reconstitution without the risk of GVHD.

Description: Acute graft-versus-host disease (aGVHD) results from alloreactive donor derived T cells attacking targets in the gastrointestinal tract, liver and skin. We observed the initiation and rapid kinetics of aGVHD in a murine model [FVB/N (H-2q) into irradiated Balb/c (H-2d)] using in vivo bioluminescence imaging. The transition from the initiation to the effector phase of aGVHD (day 3–4) was characterized by rapid T cell proliferation and upregulation of gut homing receptors alpha4beta7, alphaEbeta7 and CCR9 on alloreactive T cells in Peyer’s patches (PP), mesenteric lymph nodes (LN) and spleen, but not peripheral LNs. Therefore we asked whether the lack of specific lymphoid priming sites would lead to decreased alloreactive T cell infiltration in the gut compared to the liver and skin. Using PP deficient mice, we observed that mesenteric LN and spleen compensate for the lack of PP as alloreactive priming sites. Transplantation of PP and LN deficient mice (TNFalpha-/-) showed that the spleen alone was sufficient to cause the complete profile of aGVHD with a time course similar to that of wildtype mice. Splenectomized mice with intact secondary lymphoid organs also developed aGVHD. Strikingly, treatment of splenectomized recipients with blocking antibodies against the lymphoid homing receptors L-selectin and MAdCAM-1 prevented GVHD with 100% survival (〉120 d, p

Description: Acute graft-versus-host disease (aGVHD) is caused by alloreactive effector T cells attacking the gastrointestinal tract, liver and skin after allogeneic hematopoietic cell transplantation (aHCT). The mechanism by which alloreactive T cells target these organs and not others remains elusive. Recently, we reported that different secondary lymphoid organs (SLOs), as alloreactive priming sites, can imprint distinct homing phenotypes on evolving alloreactive effector cells in vivo. However, preventing access to selected lymphoid organs (via the use of blocking antibodies or recipient mice lacking Peyer’s patches (PP), PP and lymph nodes (LN) or spleens) did not alter the aGVHD organ manifestation. These findings not only suggested a high redundancy of SLOs as induction sites of aGVHD, but also questioned whether homing instruction of alloreactive T cells by these sites can explain the mechanism of aGVHD target organ manifestation. To test the homing instruction model we transplanted transgenic luciferase+ (luc+) FVB/N (H-2q, Thy1.1+) splenocytes into conditioned (2×400rad) Balb/c recipients (H-2d, Thy1.2+). On day+3 we isolated luc+ donor lymphocytes from peripheral LN, mesenteric LN, or spleens and transferred them into conditioned secondary allogeneic recipients. 16 hours later, bioluminescence imaging revealed that allogeneic luc+ T cells irrespective of their original priming site targeted the intestinal tract and liver. Subsequently, we compared aHCT of conditioned with non-conditioned secondary Balb/cRag−/− cγ-Chain−/− recipients. Surprisingly, we found allogeneic luc+ T cells accumulating in SLOs in non-conditioned recipients in contrast to intestinal and hepatic tissues in conditioned recipients. These in vivo findings establish that alloreactive effector cells migrate to aGVHD target tissues because of attraction to these sites rather than specific instruction by SLOs. Therefore, we propose a signal hierarchy model of alloreactive cell trafficking whereby inflammatory signal/ligand interactions dominate over organ-specific homing receptor/ligand interactions.

Description: Peripheral blood granulocytes from patients with chronic myelogenous leukemia (CML) were studied for accessibility of membrane sialic acid and galactose residues to sodium borohydride-3H radiolabeling after oxidation with sodium metaperiodate (PI/B3H4) or with galactose oxidase (GO/B3H4). Granulocytes from untreated patients with chronic myelogenous leukemia showed increased radiolabeling with PI/B3H4, and decreased labeling with GO/B3H4 when compared to normal granulocytes. Granulocytes from leukemic patients receiving chemotherapy showed normal labeling patterns. Gel electrophoresis of membrane extracts showed that the changes in PI/B3H4 and GO/B3H4 reactivity of CML cells were distributed over all membrane protein bands. Our data suggest that CML granulocyte membrane proteins are aberrantly sialylated, with decreased accessibility of galactose residues, and that these changes may be reversed by clinical drug treatment.

Description: Dyskeratosis congenita (DC) is a congenital disorder characterized by very short telomeres. Clinical presentation includes a diagnostic triad (lacey reticular pigmentation, nail dystrophy, and leukoplakia), aplastic anemia (the main cause of premature death), myelodysplastic syndrome/leukemia and solid tumors. Allogeneic HCT is the only curative option for the hematologic complications of DC but has been associated with a high risk of peri-transplant morbidity and early death. Only about fifty HCT for DC have been performed to-date, and the five year survival after related donor HCT has been about 75%, but only approximately 35% when an unrelated donor was used. To improve survival in DC patients by decreasing transplant mortality, we introduced a reduced intensity regimen including cyclophosphamide (50 mg/kg), fludarabine (200 mg/kg), low dose total body irradiation (200 cGy), and (in patients 3 and 4) campath 1H (1 mg/kg). To decrease the risk of graft rejection, grafts were not T-cell depleted. We report outcomes in four consecutive patients, two adults and two children, all of whom engrafted with donor hematopoiesis: Age (years) Sex Graft and HLA match NC dose (×108/kg) CD34 dose (×108/kg) Follow-up (months) Donor chimerism 24 M URD dUCB 4/6, 4/6 0.55, 0.39 0.5, 0.43 1 (dead*) 83% 29 F REL PBSC 6/6 13.93 4.43 20 (alive) 100% 5 F URD BM 7/8 1.38 1.55 16 (alive) 100% 2 M URD BM 8/8 5.92 2.31 3 (alive) 100% Legend: M, male; F, female; NC, nucleated cell; URD, unrelated donor; REL, related donor; BM, bone marrow; dUCB, double umbilical cord blood; PBSC, peripheral blood stem cells; *Patient had autologous recovery after the first dUCB and died of sepsis 1 month after the second dUCB; HLA matching is reported for antigen level HLA-A, B and allele level DRB1 for cord blood, and allele level typing for HLA-A, B, C, DRB1 for PBSC or BM. The most recent donor chimerism is reported. To decrease the risk of graft rejection and prevent graft versus host disease (GvHD) patients received cyclosporine and mycophenolate mofetil. Patient 2 developed limited chronic GvHD and patient 4 developed grade III skin acute GvHD. Both were treated successfully with systemic and topical steroids. Our data suggest that this conditioning regimen results in a low rate of transplant related complications without compromising engraftment. Critically, early fatal pulmonary and vascular complications, common in post-transplant courses in DC patients, were not observed. This highlights the need to avoid drugs that are associated with pulmonary toxicity such as busulfan, and to limit radiation to the lung in patients with DC. This new less intensive conditioning regimen appears to result in a low rate of transplant related complications, and yet has adequate immunosuppressive activity to permit engraftment from alternative donors in DC patients.

Description: We ascertained the prevalence of self-reported late occurrence of diabetes, hypertension, and cardiovascular (CV) disease in 1089 hematopoietic cell transplantation (HCT) survivors who underwent HCT between 1974 and 1998, survived at least 2 years, and were not currently taking immunosuppressant agents and compared them with 383 sibling controls. All subjects completed a 255-item health questionnaire. The mean age at survey completion was 39.3 years for survivors and 38.6 years for siblings; mean follow-up was 8.6 years. Adjusting for age, sex, race, and body mass index (BMI), survivors of allogeneic HCT were 3.65 times (95% confidence interval [CI], 1.82-7.32) more likely to report diabetes than siblings and 2.06 times (95% CI, 1.39-3.04) more likely to report hypertension compared with siblings but did not report other CV outcomes with any greater frequency. Recipients of autologous HCTs were no more likely than siblings to report any of the outcomes studied. Allogeneic HCT survivors were also more likely to develop hypertension (odds ratio [OR] = 2.31; 95% CI, 1.45-3.67) than autologous recipients. Total body irradiation (TBI) exposure was associated with an increased risk of diabetes (OR = 3.42; 95% CI, 1.55-7.52). Thus, HCT survivors have a higher age- and BMI-adjusted risk of diabetes and hypertension, potentially leading to a higher than expected risk of CV events with age.

Description: Abstract 2436 Poster Board II-413 Sensitive in vivo imaging methods have advanced the fields of stem cell transplantation, graft-versus–host disease (GVHD) and graft-versus-tumor responses (GVT). Near-infrared (NIF) fluorescent proteins (FP) appear advantageous for deeper tissue penetration due to minimized absorbance by hemoglobin, water and lipids. Therefore we tested whether a recently published NIF FP (FP635, “Katushka”) could serve as a single reporter for whole body and single cell imaging. To compare signal intensities of eGFP and FP635 we generated fluorescent MOSEC cell lines (mouse ovarian cancer), titrated them in vitro and subcutaneously (s.c.) in vivo in Balb/c nu/nu mice. MOSEC FP635 showed twice the signal intensities compared to MOSEC eGFP in vitro by spectral fluorescence imaging (FLI). In vivo the eGFP signal was attenuated 〉60% in contrast to only 20% for FP635 from subcutaneous sites. However, FP635 signals from deep tissue layers were quenched. To address whether reduced signal attenuation of FP635 may allow sensitive visualization of immune processes by FLI and multiphoton-laser-scanning-microscopy (MPM) we generated transgenic mice in the genetic C57Bl/6 (B6) background, expressing FP635 under the ubiquitin promoter. Transgenic founders were selected upon signal intensities of leukocyte populations measured by flow cytometry in the PerCP channel. Combination of FP635 with colors other than red were possible for multiparameter flow cytometry. Next, eGFP, DsRed and FP635 splenocytes from transgenic donors were titrated as described above. In vitro signal intensities of FP635 splenocytes were 〉5 times lower compared to the other two FPs. FP635 signal absorption in vivo was low (30%) which is consistent with MOSEC titration results. In vivo DsRed detection was most sensitive and signals were similarly attenuated as FP635 in contrast to eGFP (60%). Subsequently, we aimed to visualize FP635 in a model of GVHD, where alloreactive T cells undergo massive expansion. Balb/c nu/nu mice were lethally irradiated and transplanted with 5×106 B6.WT bone marrow cells plus either 2×107 B6.DsRed+Luciferase+ or 2×107 B6.FP635 splenocytes. Sensitivity for DsRed cell detection was superior over FP635 cells. FP635 signal was only weakly detectable in lymph nodes (LN) by ex vivo FLI, where DsRed signals were detectable at earlier timepoints and LNs were even visualized by in vivo FLI. DsRed+ Luciferase+ double transgenic splenocytes allowed direct comparison of bioluminescence imaging (BLI) to FLI. Timely in vivo visualization of immune cells in deep tissues was feasible only by BLI. After whole body imaging the suitability of FP635 for MPM was checked by co-injecting eGFP B cells and either DsRed or FP635 T cells intravenously into RAG-/- mice. As FP635 is a NIF FP we expected to achieve deeper tissue penetration in hemoglobin rich organs, such as the spleen, in single cell microscopy. After 6 weeks of adoptive cell transfer we imaged spleens by MPM. Tissue penetration depths of DsRed or FP635 T cells were compared to eGFP B cells. No advantage in penetration depth of FP635 over DsRed was measured. Photobleaching is an important factor for microscopy, especially if cells are to be tracked over long time. FP635 transfected 293T cells bleached faster (t1/2=108 sec) than 293T cells transfected with eGFP (t1/2〉900 sec) or DsRed (t1/2=411 sec). These experiments indicate that very high expression levels of FP635 need to be achieved for imaging. The signal attenuation of FP635 is low which may increase the sensitivity but in our hands DsRed showed comparable characteristics. Yet, the fast photobleaching of FP635 compared to the broadly established FPs DsRed and eGFP may be disadvantageous for long term microscopic tracking of cells. Our data indicate that BLI is by far superior over FLI in sensitivity and tissue penetration for whole body imaging of immune cells. However, FLI of red or near-infrared clonally selectable tumor cell lines may provide a welcome color addition to study immune cell-tumor interactions in combined models of BLI and FLI. Disclosures: No relevant conflicts of interest to declare.

Description: Background: High-dose chemotherapy with autologous stem cell transplantation (ASCT) is a widely used treatment strategy in lymphoma and myeloma; however, no standard approach for the mobilization of peripheral hematologic stem and progenitor cells (HSPCs) has been established. Levels of circulating CD34+ cells, a surrogate marker for mobilization efficiency, vary widely between pts, and may be influenced by disease state, prior therapy, and/or mobilization regimen. Methods: The Washington University (St. Louis, MO) transplantation database includes clinical parameters from 407 multiple myeloma (MM), 562 non-Hodgkin’s Lymphoma (NHL), and 164 Hodgkin’s disease (HD) pts who received an ASCT between 1995 and 2006. A retrospective analysis of this large (1133 pts) population was conducted to determine factors associated with mobilization efficiency. Mobilization failure was defined as collection of 〈 2 × 10^6 CD34+ cells/kg within 5 apheresis days. Statistical analysis included analysis of variance (ANOVA) with Scheffe Test to determine differences in mobilization between the various mobilization regimens (G-CSF, G-CSF/chemotherapy, G-/GM-CSF, G-CSF/AMD3100). Results: All pts were included in the analysis; 87% received G-CSF alone as the initial mobilization regimen. Mobilization failure rates are summarized in Table 1. NHL and HD pts had an approx. 4-fold higher failure rate than MM pts. The combination of G-CSF with chemotherapy increased the median CD34+ yield compared to G-CSF alone, although no obvious impact on the failure rate was noted in this relatively small group of pts. Remobilization was associated with high failure rates in NHL (79.2%), HD (77.1%), and MM (73.3%). Pooled collections were