Search

In order to maintain remission and to prolong overall-survival after allogeneic stem cell transplantation (allo-SCT) and donor lymphocyte infusion (DLI), cytotoxic T-cell responses against malignant cells play a pivotal role, however they are not well characterized so far. In this study, we focused on the detection of immune responses in patients before and after DLI. A broader epitope-specific T cell activity is associated with clinical response of patients treated with DLI and a concurrent reduction of regulatory T cell frequency may contribute to clinical response in patients after DLI. For a better characterization of the T cell responses, frequency and diversity of leukemia-associated antigen (LAA)-specific cytotoxic T cells was assessed using ELISpot and pMHC multimer assays. Furthermore, the frequency of regulatory T cells (Treg) before and after DLI was analyzed. Results were correlated to the clinical course of the patients. Independently of their clinical response, 7/11 patients (63.6%) showed an immunological response through an increase in the number of recognized epitopes over the course of DLI. There was a significant increase (p=0.02) in epitope recognition comparing early screening and maximum response after DLI and a significant increase in the mean augmentation from 1 to 4 of the spotted epitopes in the course of DLI was detected in the cohort of clinical responders (R) compared to non-responders (NR) who did not show any dynamics in epitope recognition with a median of 3 to 4 recognized LAA. The proportion of the CD4+CD25highFoxP3+ Treg within the CD4+CD25high T cell fraction decreased significantly in R from a median of 72.9% to 54.6% when comparing the variation at the time points before and after DLI (p=0.04). In NR there was no significant change in the Treg fraction in the course of DLI. In general, significantly more LAA-derived T cell epitopes (p=0.02) were recognized in R when compared to NR. Moreover, the frequency of Treg in R decreased significantly (p=0.008) while remaining stable in NR. Taken together, an increase of specific CTL responses against several LAA after DLI was detected. This study suggests that decreasing numbers of Treg as well as enhanced LAA diversity in T cell responses contribute to clinical outcome of patients treated with DLI. Disclosures Döhner: AbbVie: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Bristol Myers Squibb: Research Funding; AROG Pharmaceuticals: Research Funding; Agios: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Agios: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Pfizer: Research Funding; Seattle Genetics: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Pfizer: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celator: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding.

Clinical and preclinical data suggest that acute myeloid leukemia (AML) with mutated nucleophosmin 1(NPM1mut) may constitute an immunogenic leukemia subtype. NPM1mut AML generally correlates with a better prognosis, however the underlying mechanisms still need to be clarified. Checkpoint inhibition targeting Programmed cell death protein 1 (PD-1)/Programmed cell death 1 ligand 1 (PD-L1) has been proven to be an effective novel immunotherapeutic approach in cancer treatment including the treatment of hematological malignancies. Expression of CD34/CD38/CD274 was evaluated in 20 NPM1mut versus 20 wild-type (NPM1wt) AML patient samples via flow cytometry analyses to assess PD-L1 (CD274) expression in leukemic cells, including leukemic progenitor and stem cells (LSC). We also investigated the influence of the anti-PD-1 antibody Nivolumab® on the antigen-specific immune responses in ELISpot assays. Additionally, we assessed the effect of Nivolumab in colony forming unit (CFU) immunoassays. Many AML cases showed relevant expression of PD-L1. Bulk cells of NPM1mut AML showed a significantly higher PD-L1 expression in comparison to NPM1wtAML patients (median of 1.5%, range 0.0-8.5%, versus 0.3%, range 0.1-1.1%). Importantly, PD-L1 expression was detected at a higher level in leukemic progenitor cells (CD34+CD38-) of NPM1mut than of NPM1wtAML (median of 3.3%, range 0.0-17.2%, versus 0.3%, range 0.0-3.0%). In general, the LSC fraction showed a higher PD-L1 expression than the non-LSC fraction. CFU immunoassays showed a significant inhibition of CFU when adding T cells stimulated against various LAA. In all patient samples, effectors activated against at least one LAA were successful to decrease the colony number significantly. Immune effects increased adding Nivolumab to the CTL for several days before starting CFU immunoassays. In summary, we detected higher PD-L1 expression in NPM1mut patients, especially in the leukemic progenitor compartment. This observation further supports the hypothesis that NPM1-directed immune responses might play an important role in tumor cell rejection, which tumor cells try to escape via expression of PD-L1. Immunogenicity of neoantigens derived from NPM1mut with higher PD-L1 expression constitute promising target structures for individualized immunotherapeutic approaches. Disclosures Schrezenmeier: Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding. Bullinger:Pfizer: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Janssen: Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Sanofi: Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer Oncology: Research Funding. Döhner:Novartis: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Pfizer: Research Funding; Seattle Genetics: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Pfizer: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria.

The contribution of hepatitis C virus (HCV) infection to liver disease after bone marrow transplantation (BMT) was retrospectively evaluated in 61 patients treated with BMT. HCV genome, as well as antibodies to HCV, was analyzed in sera collected before and serially after BMT. Six patients had been infected with HCV before BMT and three patients acquired the infection during or shortly after BMT. All patients infected before BMT died within 10 weeks after transplantation. Five of these six patients (83%) died of veno-occlusive disease (VOD), compared with nine of 52 patients (17%) not infected with HCV (P.005). Risk factors for VOD other than HCV were not more prevalent in these patients compared with uninfected patients. Parallel to the development of VOD, replication of HCV increased, as demonstrated by rising concentrations of viral RNA in serum. HCV infection acquired during or after BMT caused only mild acute hepatitis C, which progressed to chronic hepatitis C in one patient surviving 10 years after BMT. These data suggest that patients with liver disease caused by HCV infection are at high risk of developing lethal VOD after BMT.

To improve the clinical outcome of patients with acute myeloid leukemia (AML), immune therapies targeting leukemia associated antigens (LAAs) might be an approach complementary to chemotherapy and transplantation of hematopoetic stem cells. The receptor for hyaluronic acid mediated motility (RHAMM/CD168) has been defined as a LAA with specific expression. To define T cell epitopes of RHAMM/CD168 towards specific T cell immunotherapies, ten peptides were synthesized considering different computer algorithms and subjected to ELISPOT assays for interferon gamma and granzyme B, and to Cr-51 release assays. CD8+ T cells taken from the peripheral blood (PB) of 13 AML patients and presensitized with the RHAMM/CD168-derived peptides R3 (ILSLELMKL) or R5 (SLEENIVIL) did specifically recognize T2 cells pulsed with R3/R5. In contrast, CD8+ T cells isolated from the PB of 21 healthy volunteers were not able to lyse R3 or R5 pulsed T2 cells, even after presensitization. COS7 cells co-transfected with HLA-A*0201 and RHAMM/CD168 were lysed by R3 or R5 presensitized CD8+ T cells. Single HLA-A*0201 or RHAMM/CD168 transfected COS7 were not recognized. Cross-reactivity of the T cells was excluded by the use of unrelated peptides. K562 cells positive for RHAMM/CD168, but lacking HLA-class I molecules were not recognized indicating T cells and not NK cells as effector cells. The HLA class-I restricted lysis of COS-7 HLA-A*0201 and RHAMM/CD168 double- transfectants was confirmed by HLA class-I blocking antibody experiments. In an AML patient having received AML blast-derived dendritic cells, a higher frequency of RHAMM/CD168-peptide specific T cells was observed after four vaccinations when compared to his T cell status before vaccination. RHAMM/CD168 is also expressed in patients with other hematological malignancies which suggests a broad clinical applicability of its newly characterized T cell epitope peptides as a potential cancer vaccine.