Predictive significance of the European LeukemiaNet classification of genetic aberrations in patients with acute myeloid leukaemia undergoing allogeneic stem cell transplantation.

OBJECTIVES: The purpose of this study was to evaluate the predictive capacity of the European LeukemiaNet (ELN) classification of genetic risk in patients with acute myeloid leukaemia (AML) undergoing allogeneic stem cell transplantation (alloSCT). METHODS: We retrospectively analysed 274 patients transplanted at our centre between 2004 and 2014. RESULTS: The ELN grouping is comparable to the Southwest Oncology Group/Eastern Cooperative Oncology Group (SWOG/ECOG) stratification in predicting the outcome after alloSCT [overall P = 0.0064 for disease-free survival (DFS), overall P = 0.003 for relapse]. Patients with an intermediate-1 profile have a significantly elevated 5-yr relapse incidence as compared to favourable risk patients, that is 40% vs. 15%, [hazard ratio (HR) 2.58, P = 0.048]. An intermediate-1 risk profile is an independent predictor for relapse as determined by multivariate Cox regression analysis (HR 3.05, P = 0.023). In intermediate-1 patients, the presence of an FLT3 internal tandem duplication (FLT3-ITD) is associated with a significantly increased relapse incidence (P = 0.0323), and a lower DFS (P = 0.0465). FLT3-ITD is an independent predictor for overall survival, DFS and relapse incidence in the intermediate-1 subgroup. CONCLUSIONS: The ELN stratification of genetic risk predicts the outcome of patients with AML undergoing alloSCT. Patients with an intermediate-1 profile have a high risk for treatment failure due to relapse, which prompts the development of alternative treatment strategies.

Severe radiotoxicity in an allogeneic transplant recipient with a heterozygous ATM mutation.

Patients receiving radiotherapy often experience toxicity of the skin and mucous membranes. While radiotherapy is a mainstay of myeloablative conditioning for allogeneic hematopoietic stem cell transplantation (ASCT), no risk factors for radiotoxicity have been identified in this setting. Here, we report on a patient with excessive radiation-induced toxicity after ASCT who carried a heterozygous mutation in the Ataxia telangiectasia mutated (ATM) gene. This is the first case to suggest a genetic basis for increased radiotoxicity after myeloablative ASCT.

Allogeneic stem cell transplantation for refractory acute myeloid leukemia: a single center analysis of long-term outcome.

For patients with refractory acute myeloid leukemia (AML), allogeneic stem cell transplantation (alloSCT) represents the only curative approach. We here analyzed the long-term outcome of 131 consecutive patients with active AML, which was either primary refractory or unresponsive to salvage chemotherapy, transplanted at our center between 1997 and 2013. After a median follow-up of 48 months for the surviving patients, disease-free survival (DFS) at 5 yr post alloSCT was 26% (94% CI: 17-35). Relapses, most of which occurred within the first 2 yr from transplant, were the predominant cause of treatment failure affecting 48% (95%CI: 40-58) of patients, whereas non-relapse mortality was 26% (95%CI: 20-36) at 5 yr and thereafter. A marrow blast count ≥20% before alloSCT was an independent prognosticator associated with an inferior DFS (HR: 1.58, P = 0.027), whereas the development of chronic graft-versus-host disease (cGvHD) predicted an improved DFS (HR 0.21, P < 0.001) and a decreased relapse incidence (HR: 0.18, P = 0.026), respectively. These results indicate that alloSCT represents a curative treatment option in a substantial proportion of patients with refractory AML. A pretransplant blast count <20% before alloSCT and the development of cGvHD are the most important predictors of long-term disease control.

Impact of early remission by induction therapy on allogeneic stem cell transplantation for acute myeloid leukemia with an intermediate-risk karyotype in first complete remission.

For patients with acute myeloid leukemia (AML) early achievement of remission during induction treatment is an important predictor for long-term outcome irrespective of the type of consolidation therapy employed. Here, we retrospectively examined the prognostic impact of early remission (ER) vs. delayed remission (DR) in a cohort of 132 AML patients with an intermediate-risk karyotype undergoing allogeneic stem cell transplantation (alloSCT) in first complete remission (CR1). In contrast to patients showing DR, patients achieving ER had a significantly higher 3-yr overall survival (OS) and disease-free survival (DFS) of 76% vs. 54% (P = 0.03) and 76% vs. 53% (P = 0.03). Likewise, 3 yr after alloSCT the cumulative incidence of relapse (CI-R) was significantly lower in the ER subgroup as compared to patients achieving DR, that is, 10% vs. 35% (P = 0.004), whereas non-relapse mortality (NRM) did not differ significantly. Multivariate analysis identified DR as an independent prognosticator for an inferior DFS (HR 3.37, P = 0.002) and a higher CI-R (HR 3.55, P = 0.002). Taken together, these data may indicate that the rapid achievement of remission predicts a favorable outcome in patients with intermediate-risk AML undergoing alloSCT in CR1. In turn, the adverse effect of DR may not be fully overcome by alloSCT.

Cytogenetic risk grouping by the monosomal karyotype classification is superior in predicting the outcome of acute myeloid leukemia undergoing allogeneic stem cell transplantation in complete remission.

We retrospectively analyzed the impact of cytogenetic abnormalities grouped according to the monosomal karyotype (MK) classification or the Southwest Oncology/Eastern Cooperative Oncology Group (SWOG/ECOG) definition in 263 patients with acute myeloid leukemia (AML) who underwent allogeneic stem cell transplantation (alloSCT) in complete remission (CR) at our center. Risk grouping using the MK criteria shows a highly significant difference in 5-yr overall survival (OS) ranging between 67%, for the most favorable, and 32%, for the poorest risk group (P = 0.001). Although similarly precise in predicting OS, the MK scheme better separates patients with respect to relapse incidence as compared to the SWOG/ECOG grouping (P = 0.0001 vs. P = 0.01). Notably, patients displaying non-MK abnormalities (MK-) had a 5-yr relapse incidence identical to those cytogenetically normal (CN), that is 24%. Multivariate analysis revealed that the MK classification is an independent prognosticator and superior in predicting OS (hazard ratios, HR 3.74, P = 0.01) and relapse incidence (HR 3.74, P = 0.005) as compared to the SWOG/ECOG criteria. Finally, subgroup analysis revealed that the prognostic capacity of the MK classification is highly significant in patients treated with standard myeloablative conditioning prior to alloSCT (P = 0.0011 for OS, P = 0.0007 for relapse). In contrast, the MK grouping failed to predict OS or relapse incidence in patients treated with reduced intensity conditioning. Taken together, these results indicate that the MK classification is superior in predicting the overall outcome of patients with AML undergoing alloSCT in CR. Furthermore, our data suggest that the genetic risk profile of MK- and CN patients is mostly overlapping in this setting.

Adoptive transfer of T-cell precursors enhances T-cell reconstitution after allogeneic hematopoietic stem cell transplantation.

Immunoincompetence after allogeneic hematopoietic stem cell transplantation (HSCT) affects in particular the T-cell lineage and is associated with an increased risk for infections, graft failure and malignant relapse. To generate large numbers of T-cell precursors for adoptive therapy, we cultured mouse hematopoietic stem cells (HSCs) in vitro on OP9 mouse stromal cells expressing the Notch-1 ligand Delta-like-1 (OP9-DL1). We infused these cells, together with T-cell-depleted mouse bone marrow or purified HSCs, into lethally irradiated allogeneic recipients and determined their effect on T-cell reconstitution after transplantation. Recipients of OP9-DL1-derived T-cell precursors showed increased thymic cellularity and substantially improved donor T-cell chimerism (versus recipients of bone marrow or HSCs only). OP9-DL1-derived T-cell precursors gave rise to host-tolerant CD4+ and CD8+ populations with normal T-cell antigen receptor repertoires, cytokine secretion and proliferative responses to antigen. Administration of OP9-DL1-derived T-cell precursors increased resistance to infection with Listeria monocytogenes and mediated significant graft-versus-tumor (GVT) activity but not graft-versus-host disease (GVHD). We conclude that the adoptive transfer of OP9-DL1-derived T-cell precursors markedly enhances T-cell reconstitution after transplantation, resulting in GVT activity without GVHD.

A phase 1/2 trial to assess safety and efficacy of a vaporized 5-methoxy-N,N-dimethyltryptamine formulation (GH001) in patients with treatment-resistant depression.

Background: Treatment-resistant depression (TRD) is a substantial public health burden, but current treatments have limited effectiveness. The aim was to investigate the safety and potential antidepressant effects of the serotonergic psychedelic drug 5-MeO-DMT in a vaporized formulation (GH001) in adult patients with TRD. Methods: The Phase 1 part (n = 8) of the trial investigated two single dose levels of GH001 (12 mg, 18 mg) with a primary endpoint of safety, and the Phase 2 part (n = 8) investigated an individualized dosing regimen (IDR) with up to three increasing doses of GH001 (6 mg, 12 mg, and 18 mg) within a single day, with a primary endpoint of efficacy, as assessed by the proportion of patients in remission (MADRS ≤ 10) on day 7. Results: Administration of GH001 via inhalation was well tolerated. The proportion of patients in remission (MADRS ≤ 10) at day 7 was 2/4 (50%) and 1/4 (25%) in the 12 mg and 18 mg groups of Phase 1, respectively, and 7/8 (87.5%) in the IDR group of Phase 2, meeting its primary endpoint (p < 0.0001). All remissions were observed from day 1, with 6/10 remissions observed from 2 h. The mean MADRS change from baseline to day 7 was -21.0 (-65%) and - 12.5 (-40%) for the 12 and 18 mg groups, respectively, and - 24.4 (-76%) for the IDR. Conclusion: Administration of GH001 to a cohort of 16 patients with TRD was well tolerated and provided potent and ultra-rapid antidepressant effects. Individualized dosing with up to three doses of GH001 on a single day was superior to single dose administration.Clinical Trial registration: Clinicaltrials.gov Identifier NCT04698603.

Absence of P-selectin in recipients of allogeneic bone marrow transplantation ameliorates experimental graft-versus-host disease.

Alloreactive T cells are crucial for graft-versus-host disease (GVHD) pathophysiology, and modulating their trafficking patterns has been efficacious in ameliorating experimental disease. We report in this paper that P-selectin, a glycoprotein found on resting and inflamed endothelium, is important for donor alloreactive T cells trafficking into GVHD target organs, such as the intestines and skin. Compared with wild-type (WT) recipients of allogeneic bone marrow transplantation, P-selectin(-/-) recipients exhibit decreased GVHD mortality and decreased GVHD of the skin, liver, and small bowels. This was associated with diminished infiltration of alloactivated T cells into the Peyer's patches and small bowels, coupled with increased numbers of donor T cells in the spleen and secondary lymphoid organs (SLOs). Surprisingly, however, donor T cells deficient for P-selectin glycoprotein ligand 1, the most well described P-selectin ligand, mediated GVHD similar to WT T cells and accumulated in SLO and target organs in similar numbers as WT T cells. This suggests that P-selectin may be required for trafficking into inflamed tissues but not SLO and that donor T cells may use multiple P-selectin ligands apart from P-selectin glycoprotein ligand 1 to interact with P-selectin and traffic into inflamed tissues during GVHD. We conclude that targeting P-selectin may be a viable strategy for GVHD prophylaxis or treatment.

A modified EBMT risk score predicts the outcome of patients with acute myeloid leukemia receiving allogeneic stem cell transplants.

The systematic and standardized pretransplant risk assessment represents an important tool to predict the outcome of patients undergoing allogeneic stem cell transplantation (alloSCT). To investigate the capacity of a modified European group for blood and marrow transplantation (mEBMT) risk score to predict the outcome of patients with acute myeloid leukemia (AML) receiving allogeneic stem cell transplants, we retrospectively analyzed 214 patients transplanted at our center between 1995 and 2008. Overall survival (OS) of the whole cohort at 1, 3, and 5 yr was 62%, 48%, and 45%, whereas the cumulative incidence of relapse or non-relapse mortality (NRM) was 26%, 33%, and 33% or 19%, 21%, and 22%. In univariate analysis, a higher mEBMT risk score was associated with an inferior OS ranging from 69% for patients with a score of 0/1 to 26% for patients with a score of 5/6 at 5 yr (P < 0.0001) and steadily increasing hazard ratios for each additional score point. Likewise, a higher mEBMT risk score was associated with an increased incidence of relapse (P = 0.049). Importantly, the prognostic value of the mEBMT risk score in terms of OS and relapse was maintained in multivariate analysis. Taken together, this indicates that a mEBMT risk score may be used to predict the outcome of patients with AML following alloSCT.

A Phase 1, Dose-Ranging Study to Assess Safety and Psychoactive Effects of a Vaporized 5-Methoxy-N, N-Dimethyltryptamine Formulation (GH001) in Healthy Volunteers.

5-Methoxy-N,N-Dimethyltryptamine (5-MeO-DMT) is a tryptamine with ultra-rapid onset and short duration of psychedelic effects. Prospective studies for other tryptamines have suggested beneficial effects on mental health outcomes. In preparation for a study in patients with depression, the present study GH001-HV-101 aimed to assess the impact of four different dose levels of a novel vaporized 5-MeO-DMT formulation (GH001) administered via inhalation as single doses of 2 (N = 4), 6 (N = 6), 12 (N = 4) and 18 mg (N = 4), and in an individualized dose escalation regimen (N = 4) on the safety, tolerability, and the dose-related psychoactive effects in healthy volunteers (N = 22). The psychedelic experience was assessed with a novel Peak Experience Scale (PES), the Mystical Experience Questionnaire (MEQ), the Ego Dissolution Inventory (EDI), the Challenging Experience Questionnaire (CEQ), and the 5-Dimensional Altered States of Consciousness Questionnaire (5D-ASC). Further aims were to assess the impact of 5-MeO-DMT on cognitive functioning, mood, and well-being. Higher doses of 5-MeO-DMT produced significant increments in the intensity of the psychedelic experience ratings as compared to the lowest 2 mg dose on all questionnaires, except the CEQ. Prominent effects were observed following single doses of 6, 12, and 18 mg on PES and MEQ ratings, while maximal effects on PES, MEQ, EDI, and 5D-ASC ratings were observed following individualized dose escalation of 5-MeO-DMT. Measures of cognition, mood, and well-being were not affected by 5-MeO-DMT. Vital signs at 1 and 3 h after administration were not affected and adverse events were generally mild and resolved spontaneously. Individualized dose escalation of 5-MeO-DMT may be preferable over single dose administration for clinical applications that aim to maximize the experience to elicit a strong therapeutic response.

A modified EBMT risk score and the hematopoietic cell transplantation-specific comorbidity index for pre-transplant risk assessment in adult acute lymphoblastic leukemia.

BACKGROUND: Disease stage is the most important prognostic parameter in allogeneic hematopoietic cell transplantation (HCT) for acute lymphoblastic leukemia, but other factors such as donor/host histocompatibility and gender combination, recipient age, performance status and comorbidities need to be considered. Several scoring systems are available to predict outcome in HCT recipients; however, their prognostic relevance in acute lymphoblastic leukemia is not well defined. DESIGN AND METHODS: In the present study we evaluated a modified EBMT risk score (mEBMT) and the HCT-specific comorbidity index (HCT-CI) in 151 adult acute lymphoblastic leukemia patients who received allogeneic HCT from 1995 until 2007 at our center. RESULTS: Disease status was first complete remission (CR1) (47%), CR>1 (21%) or no CR (32%). Overall survival (OS) at one, two and five years was 62%, 51% and 40% and non-relapse mortality (NRM) was 21%, 24% and 32%. Median mEBMT was 3 (0-6). Higher mEBMT was associated with inferior OS (hazard ratio per score unit (HR): 1.50, P<0.001), higher NRM (HR: 1.36, P=0.042) and higher relapse mortality (HR: 1.68, P<0.001). Disease stage was the predominant prognostic factor in this score. Comorbidities were present in 71% of patients with mild hepatic disease (29%), moderate pulmonary disease (28%) and infections (23%) being the most common. Median HCT-CI was 1 (0-9). In univariate analysis a trend for inferior OS (HR: 1.08, P=0.20) and higher NRM (HR: 1.14, P=0.11) with increasing HCT-CI was observed but the level of significance was not reached. In additional analyses we found that reduced Karnofsky Performance Status (KPS) was associated with inferior OS (HR: 1.34, P=0.023) and higher relapse mortality (HR: 1.71, P=0.001) when analyzed univariately. However, KPS was associated with disease stage and significance was lost in multivariate analysis. CONCLUSIONS: The mEBMT was prognostic in our patient cohort with predominant influence of disease stage, whereas a trend but no significant prognostic value was observed for the HCT-CI.

Dual mode reflectance and fluorescence confocal laser scanning microscopy for in vivo imaging melanoma progression in murine skin.

A system was designed and developed for simultaneous fluorescence and reflectance contrast in vivo confocal imaging of murine skin using 488 nm (fluorescence mode) and 830 nm (reflectance mode) laser light sources. B16 melanoma cells and B16-enhanced green fluorescent protein (EGFP) cells were inoculated intradermally into transgenic C57BL/6-TgN (ACTbEGFP) 10sb and non-transgenic C57BL/6 mice, respectively. The inoculation sites were imaged sequentially over a 20 d period. The in vivo confocal images were correlated with ex vivo conventional microscopy. The combined modality system provided single-cell resolution and adequate image registration. In fluorescence mode, B16 melanoma cells appeared as dark objects in the bright background of the GFP expressing murine cells of the C57BL/6 transgenic mouse, and the B16-EGFP melanoma cells had a bright signal within a dark background in C57BL/6 mice. In the C57BL/6 transgenic mouse, a population of fluorescent dendritic cells was observed in the vicinity of the tumor cells. The reflectance images provide a useful reference for those areas in the dermal tissues lacking a fluorescent signal. Combined reflectance/fluorescence in vivo confocal laser scanning microscopy holds significant promise for studies of tumor progression in murine skin.

Anti-leukemia T cells in AML: TNF-α⁺ CD8⁺ T cells may escape detection and possibly reflect a stage of functional impairment.

Leukemia-associated antigens such as proteinase-3 (PR3) and Wilms' tumor protein-1 (WT-1) are potential targets of T-cell responses, which can be monitored by T-cell assays within vaccination trials and after allogeneic stem cell transplantation (SCT). In chronic myeloid leukemia (CML) an aberrant cytokine profile of antigen-specific T-cells with predominant TNF-α secretion has previously been described. The aim of this study was to investigate whether these TNF-α(+)/IFN-γ(-) CD8(+) T-cells can also be observed in AML patients after SCT. Eight HLA-A2(+) AML patients at different time points after SCT were evaluated for HLA-A2-restricted CD8(+) T-cell responses against PR3, WT-1 and influenza-A using pentamer staining and different cytokine-based T-cell assays. Antigen-specific T-cell immune responses against influenza-A and PR3 were observed in 4/8 patients, WT-1-specific T-cells could be detected in 3/8 patients. Interestingly, four different cytokine secretion profiles of antigen-specific T-cells were detected: (1) IFN-γ(+)/TNF-α(+), (2) IFN-γ(+)/TNF-α(-), (3) TNF-α(+)/IFN-γ(-) and (4) IFN-γ(-)/TNF-α(-). TNF-α(+)/IFN-γ(-) CD8(+) T-cells are an interesting biological phenomenon which can obviously be observed also in AML patients. This finding has important implications for both T-cell biology and monitoring within immunotherapy trials. The functional characterization of these TNF-α(+)/IFN-γ(-) CD8(+) T-cells needs further investigations.

Reduced intensity conditioning prior to allogeneic stem cell transplantation in first complete remission is effective in patients with acute myeloid leukemia and an intermediate-risk karyotype.

To evaluate the efficacy of reduced intensity conditioning (RIC) prior to allogeneic stem cell transplantation (alloSCT) in patients with acute myeloid leukemia (AML) in first complete remission (CR1), we retrospectively analyzed the outcome of 93 consecutive patients transplanted at our institution either following RIC (n = 37) or standard myeloablative conditioning (MAC) (n = 56) between 1999 and 2007. Projected overall survival (OS) or disease-free survival (DFS) for all patients at 1, 2, and 5 years was 78 or 70%, 65 or 57%, and 61 or 53% in the RIC group versus 73 or 70%, 68 or 62%, and 56 or 54% in the standard MAC group. In the subgroup of patients with an intermediate-risk karyotype projected OS at 1, 2, and 5 years was 86, 68, and 68% following RIC (n = 21) or 75, 69, and 66% following standard MAC (n = 36). Relapse or treatment-related mortality (TRM) was 15 or 17% (RIC group) and 26 or 14% (standard MAC group). Taken together, these data suggest that RIC-alloSCT may induce stable remissions in patients with AML transplanted in CR1. In particular, patients with an intermediate-risk karyotype ineligible to transplantation following standard MAC may benefit from RIC-alloSCT in CR1 at a low TRM.

Allogeneic hematopoietic stem cell transplantation for adult acute lymphocytic leukemia.

Allogeneic hematopoietic stem cell transplantation (alloHCT) is the single most potent treatment modality to prevent relapse in adults with acute lymphocytic leukemia, but its optimal use and timing remains a matter of intense debate and research. There is general agreement that patients with clinical features of high risk for relapse should undergo alloHCT in first complete remission. However, newer studies suggest that even patients without these risk factors may benefit. Monitoring of minimal residual disease may improve risk stratification and may complement or replace conventional risk features. Prognosis in relapsed and refractory patients is dismal, and alloHCT should be performed as soon as possible. AlloHCT also offers the only reasonable chance for cure in Philadelphia chromosome-positive acute lymphocytic leukemia; the role of imatinib is not yet clearly defined. Recent developments in unrelated-donor transplantation and reduced-intensity conditioning allow the beneficial effects of alloHCT to reach a considerably wider patient population.

Organ-derived dendritic cells have differential effects on alloreactive T cells.

Dendritic cells (DCs) are considered critical for the induction of graft-versus-host disease (GVHD) after bone marrow transplantation (BMT). In addition to their priming function, dendritic cells have been shown to induce organ-tropism through induction of specific homing molecules on T cells. Using adoptive transfer of CFSE-labeled cells, we first demonstrated that alloreactive T cells differentially up-regulate specific homing molecules in vivo. Host-type dendritic cells from the GVHD target organs liver and spleen or skin- and gut-draining lymph nodes effectively primed naive allogeneic T cells in vitro with the exception of liver-derived dendritic cells, which showed less stimulatory capacity. Gut-derived dendritic cells induced alloreactive donor T cells with a gut-homing phenotype that caused increased GVHD mortality and morbidity compared with T cells stimulated with dendritic cells from spleen, liver, and peripheral lymph nodes in an MHC-mismatched murine BMT model. However, in vivo analysis demonstrated that the in vitro imprinting of homing molecules on alloreactive T cells was only transient. In conclusion, organ-derived dendritic cells can efficiently induce specific homing molecules on alloreactive T cells. A gut-homing phenotype correlates with increased GVHD mortality and morbidity after murine BMT, underlining the importance of the gut in the pathophysiology of GVHD.

IFN-gamma and Fas ligand are required for graft-versus-tumor activity against renal cell carcinoma in the absence of lethal graft-versus-host disease.

To determine the mechanisms of graft-versus-tumor (GVT) activity in the absence of graft-versus-host disease (GVHD) against a solid tumor, we established two allogeneic bone marrow transplantation models with a murine renal cell carcinoma (RENCA). The addition of 0.3 x 10(6) donor CD8(+) T cells to the allograft increased the survival of tumor-bearing mice without causing GVHD. The analysis of CD8(+) T cells deficient in cytotoxic molecules demonstrated that anti-RENCA activity is dependent on IFN-gamma and Fas ligand (FasL), but does not require soluble or membrane-bound TNF-alpha, perforin, or TRAIL. Recipients of IFN-gamma(-/-) CD8(+) T cells are unable to reject RENCA compared with recipients of wild-type CD8(+) T cells and, importantly, neither group develops severe GVHD. IFN-gamma(-/-) CD8(+) T cells derived from transplanted mice are less able to kill RENCA cells in vitro, while pretreatment of RENCA cells with IFN-gamma enhances class I and FasL expression and rescues the lytic capacity of IFN-gamma(-/-) CD8(+) T cells. These results demonstrate that the addition of low numbers of selected donor CD8(+) T cells to the allograft can mediate GVT activity without lethal GVHD against murine renal cell carcinoma, and this GVT activity is dependent on IFN-gamma and FasL.

Absence of beta7 integrin results in less graft-versus-host disease because of decreased homing of alloreactive T cells to intestine.

The alpha4beta7 integrin plays a central role in the homing of T cells to the gut. We hypothesized that absence of the beta7 subunit would result in a reduction of intestinal graft-versus-host disease (GVHD) and an improvement in overall GVHD morbidity and mortality in recipients of hematopoietic stem cell transplantation (HSCT). Analysis of alloreactive beta7-/- T cells showed intact activation, proliferation, cytokine production, and cytotoxicity. However, recipients of beta7-/- donor T cells in murine HSCT models experienced less GVHD morbidity and mortality than recipients of wild-type (WT) T cells, associated with a decrease in donor T-cell infiltration of the liver and intestine and with an overall significant decrease in hepatic and intestinal GVHD. In graft-versus-tumor (GVT) experiments, we demonstrated intact or even enhanced GVT activity of beta7-/- donor T cells. In conclusion, beta7-/- donor T cells caused less GVHD morbidity and mortality than WT donor T cells because of selectively decreased T-cell infiltration of the liver and intestines. Our data suggest that strategies to target the beta7 integrin have the clinical potential to alleviate or prevent GVHD while sparing or potentiating GVT activity.

Glucocorticoid-induced TNF receptor family related gene activation overcomes tolerance/ignorance to melanoma differentiation antigens and enhances antitumor immunity.

Glucocorticoid-induced TNF receptor family related protein (GITR) is present on many different cell types. Previous studies have shown that in vivo administration of an anti-GITR agonist mAb (DTA-1) inhibits regulatory T cells (Treg)-dependent suppression and enhances T cell responses. In this study, we show that administration of DTA-1 induces >85% tumor rejection in mice challenged with B16 melanoma. Rejection requires CD4+, CD8+, and NK1.1+ cells and is dependent on IFN-gamma and Fas ligand and independent of perforin. Depletion of Treg via anti-CD25 treatment does not induce B16 rejection, whereas 100% of the mice depleted of CD25+ cells and treated with DTA-1 reject tumors, indicating a predominant role of GITR on effector T cell costimulation rather than on Treg modulation. T cells isolated from DTA-1-treated mice challenged with B16 are specific against B16 and several melanoma differentiation Ags. These mice develop memory against B16, and a small proportion of them develop mild hypopigmentation. Consistent with previous studies showing that GITR stimulation increases Treg proliferation in vitro, we found in our model that GITR stimulation expanded the absolute number of FoxP3+ cells in vivo. Thus, we conclude that overall, GITR stimulation overcomes self-tolerance/ignorance and enhances T cell-mediated antitumor activity with minimal autoimmunity.

DNA immunization against tissue-restricted antigens enhances tumor immunity after allogeneic hemopoietic stem cell transplantation.

Malignant relapse remains a major problem for recipients of allogeneic hemopoietic stem cell transplantation (HSCT). We hypothesized that immunization of allogeneic HSCT recipients against tissue-restricted Ags using DNA vaccines would decrease the risk of relapse without enhancing graft-vs-host disease (GVHD). Using the mouse B16 melanoma model, we found that post-HSCT DNA immunization against a single tumor Ag induces tumor rejection that is significantly greater than HSCT alone in a T cell-depleted MHC-matched minor Ag-mismatched allogeneic HSCT model (LP --> B6). In treatment models, post-HSCT DNA immunization provides significantly greater overall survival than the vaccine alone. Donor leukocyte infusion further enhances tumor-free survival, including in treatment models. There was no GVHD in HSCT recipients treated with DNA vaccination and donor leukocyte infusion. Further analysis demonstrated that these effects are dependent on CD8+ T cells of donor origin that recognize multiple epitopes. These results demonstrate that DNA immunization against tissue-restricted Ags after allogeneic T cell-depleted HSCT can induce potent antitumor effects without causing GVHD.