ALBERT

Description: AC133 is one of a new panel of murine hybridoma lines producing monoclonal IgG antibodies (mAbs) to a novel stem cell glycoprotein antigen with a molecular weight of 120 kD. AC133 antigen is selectively expressed on CD34bright hematopoietic stem and progenitor cells (progenitors) derived from human fetal liver and bone marrow, and blood. It is not detectable on other blood cells, cultured human umbilical vein endothelial cells (HUVECs), fibroblast cell lines, or the myeloid leukemia cell line KG1a by standard flow cytometric procedures. All of the noncommitted CD34+ cell population, as well as the majority of CD34+ cells committed to the granulocytic/monocytic pathway, are stained with AC133 antibody. In vitro clonogenicity assays have demonstrated that the CD34+AC133+ double-positive population from adult bone marrow contains the majority of the CFU-GM, a proportion of the CFU-Mix, and a minor population of BFU-E. The CD34dim and AC133− population has been shown to contain the remaining progenitor cells. AC133-selected cells engraft successfully in a fetal sheep transplantation model, and human cells harvested from chimeric fetal sheep bone marrow have been shown to successfully engraft secondary recipients, providing evidence for the long-term repopulating potential of AC133+ cells. A cDNA coding for AC133 antigen has been isolated, which codes for a polypeptide consisting of 865 amino acids (aa) with a predicted size of 97 kD. This antigen is modeled as a 5-transmembrane molecule, a structure that is novel among known cell surface structures. AC133 antibody provides an alternative to CD34 for the selection and characterization of cells necessary for both short- and long-term engraftment, in transplant situations, for studies of ex vivo expansion strategies, and for gene therapy.

Description: Abstract 1804 Introduction: Advanced stage follicular lymphoma (FL) cannot be cured by current conventional therapies. Treatment with idiotype cancer vaccines has thus far been disappointing, and is expensive and labor intensive. Hence, simpler principles for vaccinations that do not require production of patient-specific tumor antigens are warranted. We designed a novel strategy involving intratumoral injections of low-dose anti-CD20 antibody (Rituximab) and autologous dendritic cells (DC) in irradiated lymph nodes. The aim was to facilitate antigen up-take and presentation by DCs to T cells to achieve systemic anti-tumor responses in patients with FL. Method: Ten untreated patients with stage III/IV follicular lymphoma not in need of conventional therapy underwent standard work-up, including CT-scans, FDG PET/CT and bone marrow samples. Single tumor cells were frozen for later immunoassays. Apheresis was performed for isolation of monocytes subsequently cultured with IL-4 and GM-CSF for 5 days to generate immature dendritic cells (DC). The first 6 patients received a single 8 Gy dose of radiotherapy against an enlarged node on day 1, followed by intratumoral injections of DCs (1 × 108) and GM-CSF (50 μg) on days 3 and 4. The treatment was repeated against a different enlarged node after 6 weeks. Thereafter, the protocol was changed, and patients received intratumoral injections of a small dose of rituximab (5 mg) on days 1 and 3 in order to enhance ADCC, radiotherapy (8 Gy) on day 2 and intratumoral injections of DCs and GM-CSF sc on days 4 and 5. This therapy was then repeated after 2 and 4 weeks, targeting other lymph nodes. Follow-up included repeated PET/CT-scans, bone marrow sampling and preparation of peripheral blood mononuclear cells (PBMC) for later immune studies. Tumor-specific helper and cytotoxic T cell responses were monitored by flow cytometry after a 5-day co-culture of single tumor cells with autologous PBMC sampled before, and 2 and 4 months after, treatment. Proliferation was studied by incorporation of CFSE, whereas degranulation (CD107a/b) and interferon gamma release was measured following re-stimulation with tumor cells for 5h. Result: Ten patients with FL of a total of 20 planned for this study were treated so far. No adverse events were observed. Among the first 6 patients treated by the original strategy, two showed a systemic metabolic response by PET/CT and one of these had a good response by regular CT not qualifying for PR. The protocol was then modified as described above. The first patient treated by the new strategy that included intratumoral rituximab, had a systemic metabolic response by PET/CT after 2 and 4 months and was PET negative with a normal CT by 8 months (Figure 1). Another patient showed a systemic PET response at 2 and 4 months. Analysis of T cell responses against autologous tumor cells has been initiated and we have so far demonstrated a vigorous CD8 dominated T cell response, as assayed by proliferation, degranulation and cytokine production, in the patient who achieved a negative PET-scan at 8 months (Figure 2). In parallel with the metabolic response, the immune response became stronger during follow-up after treatment. One patient without clinical response was also analysed, and no immune response was demonstrated. Further analysis of immune responses in the other patients is on-going. Conclusion: The present strategy is a novel principle to generate T cell responses and clinical anti-tumor responses detected by PET/CT in FL. Disclosures: No relevant conflicts of interest to declare.

Description: Global gene expression profiling of the tumor microenvironment in diffuse large B-cell lymphoma (DLBCL) has revealed broad innate immune signatures that distinguish the heterogeneous disease subtypes and correlate with good treatment outcome. However, we still lack tools to identify the relatively large group of patients that are refractory to initial therapy and have a dismal prognosis. Here, we used mass cytometry and serum profiling in a systems-level approach to analyze immune responses in 36 patients with aggressive B cell lymphoma and age- and sex-matched healthy controls. Stochastic neighbor embedding (t-SNE) analysis of protein profiles divided patients into two distinct clusters, with cluster 2 representing patients with a more severe deviation in their protein expression compared to healthy controls. Patients in cluster 2 showed a more dramatic perturbation of their immune cell repertoires with expansion of myeloid-derived suppressor cells (MDSCs), increased T cell differentiation and significantly higher expression of metabolic markers such as GLUT-1 and activation markers, including Ki67, CD38 and PD-1. An extended analysis of serum protein profiles in two independent cohorts (n=69 and n=80 patients, respectively) revealed that that the identified systemic immune signatures were linked to poor progression free survival (PFS) and inferior overall survival (OS). Immune monitoring during chemo-immunotherapy showed that most patients normalized their serum protein profiles. Notably, non-responding patients retained higher than normal expression of several proteins, including PD-L1, CD70, IL-18, granzyme A and CD83. These studies demonstrate distinct patterns of disease-driven alterations in the systemic immune response of DLBCL patients that are associated with poor survival and persist in patients who are refractory to therapy. Figure 1 System-level immune signatures associated with poor prognosis in DLBCL. A) Altered serum profiles in patients compared to healthy controls. Two clusters of patients were identified based on t-SNE analysis of serum profiles. B) Patients in cluster 2 had bulky disease and B symptoms. C) t-SNE map of all patients (n=36) and controls (n=17). Relative abundance of cells from healthy controls and patients in all areas of the t-SNE clustering, highlighting cell subsets that are larger or smaller in patients compared to healthy donors. Colors indicate the difference in kernel density estimation of the t-SNE data for patients and healthy controls. D) Abundance of monocytic myeloid-derived suppressor cells as percentage of all CD45+ cells in healthy donors and the two patient clusters. White, Healthy controls; Blue, Cluster 1; Red, Cluster 2. E) Major phenotypic differences between patient clusters shown as mean mass intensity (MMI) or percent positive cells for selected markers (CD38 and PD-1) across multiple subsets. White, Healthy controls; Blue, Cluster 1; Red, Cluster 2. F-G) Overall survival in patients with serologically defined immune signatures belonging to cluster 1 or 2. H) Abundance of serum proteins in patients that stayed in remission (n=24) compared to those that did not (n=6). Figure 1 Disclosures Olweus: Gilead Kite: Research Funding; Intellia: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Wahlin:Roche and Gilead: Consultancy. Fehniger:Cyto-Sen Therapeutics: Consultancy; Horizon Pharma PLC: Other: Consultancy (Spouse). Holte:Novartis: Honoraria, Other: Advisory board. Kolstad:Nordic Nanovector: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding. Malmberg:Fate Therapeutics, Inc.: Consultancy, Research Funding; Vycellix: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Description: AC133 is one of a new panel of murine hybridoma lines producing monoclonal IgG antibodies (mAbs) to a novel stem cell glycoprotein antigen with a molecular weight of 120 kD. AC133 antigen is selectively expressed on CD34bright hematopoietic stem and progenitor cells (progenitors) derived from human fetal liver and bone marrow, and blood. It is not detectable on other blood cells, cultured human umbilical vein endothelial cells (HUVECs), fibroblast cell lines, or the myeloid leukemia cell line KG1a by standard flow cytometric procedures. All of the noncommitted CD34+ cell population, as well as the majority of CD34+ cells committed to the granulocytic/monocytic pathway, are stained with AC133 antibody. In vitro clonogenicity assays have demonstrated that the CD34+AC133+ double-positive population from adult bone marrow contains the majority of the CFU-GM, a proportion of the CFU-Mix, and a minor population of BFU-E. The CD34dim and AC133− population has been shown to contain the remaining progenitor cells. AC133-selected cells engraft successfully in a fetal sheep transplantation model, and human cells harvested from chimeric fetal sheep bone marrow have been shown to successfully engraft secondary recipients, providing evidence for the long-term repopulating potential of AC133+ cells. A cDNA coding for AC133 antigen has been isolated, which codes for a polypeptide consisting of 865 amino acids (aa) with a predicted size of 97 kD. This antigen is modeled as a 5-transmembrane molecule, a structure that is novel among known cell surface structures. AC133 antibody provides an alternative to CD34 for the selection and characterization of cells necessary for both short- and long-term engraftment, in transplant situations, for studies of ex vivo expansion strategies, and for gene therapy.

Description: Immune responses to established cancer are difficult to evoke by vaccination of patients due to various mechanisms of tolerance. In contrast, T cells in allogeneic stem cell grafts can contribute to cure of a number of hematological malignancies by the graft-versus-leukemia (GvL) effect. However, unknown T cell specificities causing life-threatening graft-versus-host disease (GvHD) limit the applicability of this treatment. Selection of GvL activity would increase the efficiency, safety and applicability of allogeneic stem cell transplantation. Here, we describe a novel method for overcoming tolerance by generation of allo-restricted, antigen-specific T cells. These cells specifically recognize and kill melanoma tumor cells presenting the melanoma-associated MART-1 in context of the prevalent major histocompatibility complex (MHC) class I molecule HLA-A*0201. HLA-A*0201-negative peripheral blood mononuclear cells were co-cultured with autologous, monocyte-derived dendritic cells transfected with HLA-A*0201 mRNA and subsequently pulsed with the peptide ELAGIGILTV from the melanoma-associated antigen MART-1. Antigen-specific T cells were detected in 8/10 donors on day 12 using peptide-MHC-pentamers (median 0.28% of CD8+ T cells, range 0-4.17%), and in all donors by day 19. When needed, cells were restimulated on day 12 with HLA-A*0201 mRNA-transfected and peptide pulsed autologous EBV-transformed lymphoblastoid cell lines (EBV-LCL). MART-1 pentamer+ T cells from five donors were sorted and expanded. The cytotoxic T cell lines (CTLs) responded with production of interferon-γ, degranulation and lysis of target cells when stimulated with peptide-pulsed EBV-LCL, even at low peptide concentrations (10−5 M). Similar responses were seen to melanoma cell lines expressing HLA-A*0201 and MART-1 (FM-57, Malme3M). In contrast, MART-1+ melanoma cells lacking HLA-A*0201 (Mel202) induced responses only when transfected with HLA-A*0201. Starting out with 0.5×106 pentamer+ T cells, a potential number of 3.0×109 pentamer+ T cells could be reached four weeks later, representing a 7800-fold expansion Our data show that allo-restricted T cells with high specificity for tumor-associated antigens can be rapidly generated, sorted and expanded to clinically applicable numbers. Figure Figure

Description: Dendritic cells (DCs) are believed to regulate T cell-mediated immunity primarily by directing differentiation of naive T cells. Here, we show that a large fraction of CD4+ memory cells produce IL-10 within the first hours after interaction with plasmacytoid DCs (PDCs). In contrast, CD11c+ DCs induce IFN-γ and little IL-10. IL-10–secreting T cells isolated after 36 hours of culture with PDCs suppressed antigen-induced T-cell proliferation by an IL-10–dependent mechanism, but were distinct from natural and type 1 regulatory T cells. They proliferated strongly and continued to secrete IL-10 during expansion with PDCs, and after restimulation with immature monocyte-derived DCs or CD11c+ DCs. The IL-10–producing T cells acquired the ability to secrete high levels of IFN-γ after isolation and subsequent coculture with PDCs or CD11c+ DCs. Compared to CD11c+ DCs, PDCs were superior in their ability to selectively expand T cells that produced cytokines on repeated antigenic challenge. The DC-dependent differences in cytokine profiles were observed with viral recall antigen or staphylococcal enterotoxin B and were independent of extracellular type I interferon or IL-10. Our results show that DCs can regulate memory responses and that PDCs rapidly induce regulatory cytokines in effector T cells that can suppress bystander activity.

Description: It has been suggested that human cytomegalovirus (HCMV) evades the immune system by infecting and paralyzing antigen-presenting cells. This view is based mainly on studies of dendritic cells (DCs) obtained after culture of monocytes (moDCs). It is contradicted by the asymptomatic course of HCMV infection in healthy persons, indicating that other key antigen-presenting cells induce an efficient immune response. Here we show that HCMV activates CD11c+ DCs and plasmacytoid DCs (PDCs). In contrast to moDCs, CD11c+ DCs and PDCs produced interferon (IFN) type 1 when exposed to HCMV. Autocrine IFN type 1 partially protected CD11c+ DCs against infection, whereas PDCs were resistant to HCMV even when IFN type 1 activity was inhibited. HCMV exposure induced the maturation of CD11c+ DCs by IFN type 1-dependent and -independent mechanisms. Importantly, CD11c+ DCs infected by inhibiting IFN type 1 activity retained full capacity to stimulate T cells. Renal transplant recipients receiving immunosuppressive treatment had lower frequencies of CD11c+ DCs and PDCs in blood than did healthy controls. The results show that HCMV activates the immune system by interacting with CD11c+ DCs and PDCs and that recipients of renal transplants have low frequencies of these cell types in blood.