Gabriele Köhler

Phase I study in melanoma patients of a vaccine with peptide‐pulsed dendritic cells generated in vitro from CD34+ hematopoietic progenitor cells

Dendritic cells (DCs) are professional antigen‐presenting cells (APCs) that can be used for vaccination purposes, to induce a specific T‐cell response in vivo against melanoma‐associated antigens. We have shown that the sequential use of early‐acting hematopoietic growth factors, stem cell factor, IL‐3 and IL‐6, followed by differentiation with IL‐4 and granulocyte‐macrophage colony‐stimulating factor allows the in vitro generation of large numbers of immature DCs from CD34+ peripheral blood progenitor cells. Maturation to interdigitating DCs could specifically be induced within 24 hr by addition of TNF‐α. Here, we report on a phase I clinical vaccination trial in melanoma patients using peptide‐pulsed DCs. Fourteen HLA‐A1+ or HLA‐A2+ patients received at least 4 i.v. infusions of 5 × 106 to 5 × 107 DCs pulsed with a pool of peptides including either MAGE‐1, MAGE‐3 (HLA‐A1) or Melan‐A, gp100, tyrosinase (HLA‐A2), depending on the HLA haplotype. A total of 83 vaccinations were performed. Clinical side effects were mild and consisted of low‐grade fever (WHO grade I–II). Clinical and immunological responses consisted of anti‐tumor responses in 2 patients, increased melanoma peptide‐specific delayed‐type hypersensitivity reactions in 4 patients, significant expansion of Melan‐A‐ and gp100‐specific cytotoxic T lymphocytes in the peripheral blood lymphocytes of 1 patient after vaccination and development of vitiligo in another HLA‐A2+ patient. Our data indicate that the vaccination of peptide‐pulsed DCs is capable of inducing clinical and systemic tumor‐specific immune responses without provoking major side effects. Int. J. Cancer 86:385–392, 2000.

A phase‐I clinical study of autologous tumor cells plus interleukin‐2‐gene‐transfected allogeneic fibroblasts as a vaccine in patients with cancer

Tumor cells transfected to express immunostimulatory cytokines, or admixed with similarly modified bystander cells, are able to induce immune responses against unmodified tumor cells in animal models. For treatment of human patients, a vaccine composed of autologous tumor cells and IL‐2‐secreting allogeneic fibroblasts was developed. Autologous tumor cells were isolated from biopsy specimens. A clone (KMST6.14) of an immortalized human fibroblast line that stably secreted 5290 IU IL‐2 per 106 cells and per 24 hr was obtained by cationic lipofection with an expression construct for human IL‐2 and Neor. Fifteen patients with refractory malignant tumors received 3–4 injections of irradiated KMST6.14 and autologous tumor cells in a phase‐I clinical trial. Increasing transient inflammatory responses without systemic toxicity developed at vaccination sites and after injections with irradiated tumor cells only (p < 0.05). These sites contained a dense infiltrate of CD3+ T cells with numbers of CD4+ helper cells exceeding those of CD8+ cytotoxic T cells (CTL). CD8+ T‐cell lines isolated from vaccination sites of 2 malignant melanoma patients but not of renal‐cell carcinoma patients exhibited a dominant lytic activity against autologous tumor cells in vitro. CD8+ T‐cell clones established from the vaccination site of 1 of 2 renal‐cell carcinoma patients preferentially lysed autologous and partially matched allogeneic renal‐cell carcinoma cells. In conclusion, a vaccine composed of IL‐2 gene‐transfected allogeneic fibroblasts and autologous tumor cells is able to enhance specific anti‐tumor T‐cell responses in vivo without major side‐effects. Malignant melanoma and renal‐cell carcinoma appear to be promising entities for testing of similar approaches in future therapeutic trials. Int. J. Cancer, 70:269–277, 1997.

CD34+ peripheral blood progenitor cell and monocyte derived dendritic cells: a comparative analysis

Dendritic cells (DC) have been generated in vitro from either CD34+ haemopoietic progenitor cells (HPC) or peripheral blood monocytes (Mo) in the presence of specific cytokine combinations, including granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). Since differences between DC from either source may be important for the clinical use of these antigen‐presenting cells (APC), a comparative analysis was performed. HPC were expanded in the presence of interleukin (IL)‐3, IL‐6 and stem cell factor (SCF) (days 1–7) and subsequently induced by IL‐4u2003+u2003GM‐CSF (days 8–26) to differentiate to Langerhans‐type cells (pLC). The latter cytokines were similarly used to generate Mo‐derived LC (mLC). Maturation of both cell types, pLC and mLC, to interdigitating DC‐type cells (iDC) was induced by tumour necrosis factor‐α (TNF‐α) or lipopolysaccharide (LPS). Analysis of mLC/pLC and miDC/piDC with respect to morphology, phenotype, antigen uptake and presentation revealed a high similarity of DC from either source. The majority of mLC, however, exhibited a more mature differentiation stage, compared to pLC, evidenced from lower numbers of multilaminar MHC class II compartments and less efficient APC function for extracellular protein antigens. Although macropinocytosis was performed by LC, neither LC nor iDC from either source were able to take up 0.5u2003μm latex beads. However, phagocytosis of 0.5u2003μm and 1u2003μm beads was performed by Mo that could subsequently be induced to become iDC, thus providing the unique opportunity to present phagocytosed material in DC‐type fashion. Mo may be the preferential source for clinical use of iDC‐type cells since preparation and culture are easier to perform and are less costly while APC function is similar to HPC‐derived iDC.

GM‐CSF promotes differentiation of a precursor cell of monocytes and Langerhans‐type dendritic cells from CD34+ haemopoietic progenitor cells

Epithelia‐associated dendritic cells (DC) including Langerhans cells in the skin (LC) are precursors of lymph node located interdigitating DC (iDC). CD1a+ LC are known to be derived from CD34+ haemopoietic progenitor cells (HPC); however, cells of an intermediate differentiation state that are CD34− and CD1a− have not been identified. Monitoring the differentiation pathway of HPC in the presence of GM‐CSFu2003+u2003IL‐4, we observed the emergence of a distinct LC precursor population that was CD33+ CD13+ CD4+ CD38+ CD44+ CD34− CD14− CD1a−. The cells could be separated by FACS due to a unique CD44/CD38 expression pattern or by CD44 expression in conjunction with the SSC profile. It was found that they were similarly generated in the presence of GM‐CSF alone and were detectable in culture for at least a week. Irrespective of being generated in the presence of GM‐CSFu2003+u2003IL‐4 or GM‐CSF alone, CD44/SSC‐sorted precursor cells matured to MHC class II compartments (MIIC) and Birbeck granules (BG) expressing LC, when subsequently cultured in the presence of GM‐CSFu2003+u2003IL‐4. When IL‐4 was omitted, however, the same cells matured to phagocytically active adherent macrophages (MΦ). These culture conditions were associated with a >u20034‐fold increase in the concentration of IL‐6 when compared to those used for LC differentiation. The identification of a distinct oligopotent precursor cell population that can deliberately be induced to give rise to BG+ MIIC+ CD1a+ CD14− LC or to adherent CD14+ MΦ further substantiates the close relationship of monocytes and DC and may help to identify its in vivo equivalent.

Reticulated platelet counts correlate with treatment response in patients with idiopathic thrombocytopenic purpura and help identify the complex causes of thrombocytopenia in patients after allogeneic hematopoietic stem cell transplantation

In thrombocytopenic conditions of unknown origin, quantification of reticulated platelets (RP) in the peripheral blood by flow cytometry has been shown to differentiate increased platelet (Plt) turnover from insufficient Plt production.

Drug resistance of secondary acute myeloid leukemia with megakaryoblastic features and p190 BCR-ABL rearrangement

A 46-year-old female presented with acute myeloid leukemia during complete remission of multiple myeloma after extensive treatment with alkylating agents. Leukemic blasts expressed CD34, platelet esterase and gp IIIa. RT-PCR analyses of peripheral blood cells detected a p190 type BCR-ABL rearrangement and high levels of MDR1. The patient expired during neutropenia shortly after induction chemotherapy. Autopsy revealed persistent blasts in the bone marrow, spleen and liver. Secondary acute myeloid leukemia with megakaryoblastic features and p190-type BCR-ABL rearrangement has not previously been reported. The possibility that the combination of a BCR-ABL rearrangement with overexpression of MDR1 may have contributed to the treatment-refractory course is discussed.