S. Van Gool

Surgery and adjuvant dendritic cell-based tumour vaccination for patients with relapsed malignant glioma, a feasibility study.

Patients with relapsed malignant glioma have a poor prognosis. We developed a strategy of vaccination using autologous mature dendritic cells loaded with autologous tumour homogenate. In total, 12 patients with a median age of 36 years (range: 11–78) were treated. All had relapsing malignant glioma. After surgery, vaccines were given at weeks 1 and 3, and later every 4 weeks. A median of 5 (range: 2–7) vaccines was given. There were no serious adverse events except in one patient with gross residual tumour prior to vaccination, who repetitively developed vaccine-related peritumoral oedema. Minor toxicities were recorded in four out of 12 patients. In six patients with postoperative residual tumour, vaccination induced one stable disease during 8 weeks, and one partial response. Two of six patients with complete resection are in CCR for 3 years. Tumour vaccination for patients with relapsed malignant glioma is feasible and likely beneficial for patients with minimal residual tumour burden.

CD40 triggering of chronic lymphocytic leukemia B cells results in efficient alloantigen presentation and cytotoxic T lymphocyte induction by up-regulation of CD80 and CD86 costimulatory molecules

Freshly collected chronic lymphocytic leukemia B cells (B-CLL cells) are known to be inefficient at stimulating allogeneic T cells, and to lack significant expression of B7 (CD80 and CD86) costimulatory molecules. We investigated the potential of CD40 triggering to up-regulate the expression of adhesion and costimulatory molecules on B-CLL cells, and to enhance their immunogenicity towards allogeneic T cells. B-CLL cells cocultured with human CD40 ligand-expressing mouse fibroblasts rapidly up-regulated CD54 and CD58 adhesion molecules and B7-1 (CD80) and B7-2 (CD86) costimulatory molecules, and acquired a strong stimulatory capacity towards CD4+ as well as isolated CD8+ allogeneic T cells. Costimulation by both CD80 and CD86 proved critical for allogeneic T cell proliferation and CD25 and HLA-DR expression, since these were strongly inhibited by anti-CD80 or anti-CD86 monoclonal antibodies, and completely abrogated by CTLA4-Ig fusion protein, which blocks both CD80 and CD86. B7 costimulation also proved critical for restimulation of primed B-CLL-reactive T cells. Most importantly, priming of purified CD8+ T cells with CD40-triggered allogeneic B-CLL cells resulted in cytotoxic activity against the unstimulated B-CLL cells. These findings raise the possibility that CD40 triggering of B-CLL cells might be exploited in immunotherapeutic protocols.

Phenotype, cytokine production and cytolytic capacity of fresh (uncultured) tumour-infiltrating T lymphocytes in human renal cell carcinoma

We investigated the phenotype and functional capacities of tumour‐infiltrating lymphocytes (TIL), freshly isolated from primary renal cell carcinoma (RCC) specimens (n = 20). Three‐colour flow cytometry immunophenotyping revealed that RCC TIL consist mainly of CD3+ T cells, with a clear predominance of CD4−CD8+ over CD4+ CD8− T cells, and a marked population of CD4+ CD8+ T cells. Natural killer (NK) cells were also strongly represented (> 25% in 15 of 20 tumour samples), while B cells constituted a minor TIL subset (< 5% in 18 of 20 tumour samples). More importantly, the T and NK cells within the tumour displayed a significantly higher expression of the early activation marker CD69 than their counterparts in adjacent normal renal tissue and in peripheral blood. Expression of CD54 and of HLA‐DR was also elevated on CD3+ TIL, and HLA‐DR expression was further vigorously up‐regulated following ex vivo stimulation with anti‐CD3, all suggesting enhanced immune activity within the tumour microenvironment. CD3+ CD4+ TIL displayed a normal capacity to up‐regulate CD25 expression and to secrete both Th1‐type (IL‐2, tumour necrosis factor‐alpha (TNF‐α) and interferon‐gamma (IFN‐γ)) and Th2‐type (IL‐4, IL‐5 and IL‐10) cytokines upon triggering with anti‐CD3. Furthermore, cytokine production was susceptible to modulation by CD28 costimulation. CD3+ CD8+ TIL, on the other hand, consistently demonstrated a poor up‐regulation of CD25 upon triggering with anti‐CD3, and displayed poor ex vivo cytolytic activity in an anti‐CD3‐redirected 4‐h cytotoxicity assay against murine P815 cells. Collectively, our findings indicate that the CD3+ CD4+ TIL in RCC have normal functional capacities, whereas the proportionally major CD3+ CD8+ TIL are functionally impaired. The relevance of these findings to the in vivo local immune response in RCC is discussed.

CD57+/CD28- T cells in untreated hemato-oncological patients are expanded and display a Th1-type cytokine secretion profile, ex vivo cytolytic activity and enhanced tendency to apoptosis

Three-color flow cytometry immunophenotyping revealed significant increases of CD57+ and CD28− cells among both circulating CD4+ and CD8+ T lymphocytes of untreated hemato-oncological patients (n = 54) as compared to healthy donors (n = 55), with CD57 and CD28 expression on the patients’ T cells being largely reciprocal. Marked expansion of CD57+ cells among circulating CD4+ T lymphocytes was frequently detected in patients with chronic leukemia of B cell origin (B-CLL, hairy cell leukemia) but not in patients with chronic myeloid leukemia, suggesting a causal relation with the tumor’s major histocompatibility complex class II expression. Using immunomagnetic separation techniques, we further demonstrate that the patients’ CD57+/CD28− T cells display a typical Th1-type cytokine secretion profile upon anti-CD3 stimulation, with a markedly higher secretion of the Th1-type cytokines IL-2, IFN-γ, and TNF-α than their CD57−/CD28+ counterparts. Cytotoxic activity of circulating CD8+ T lymphocytes, measured ex vivo in an anti-CD3-redirected assay, was almost exclusively exerted by the CD57+/CD28− subset. Moreover, a marked cytotoxic activity was detected within CD4+CD57+ T cells from some B-CLL patients. Finally, the patients’ CD57+/CD28− T cells displayed an increased tendency to apoptosis in culture. Collectively, our results indicate that the expanded CD57+/CD28− T cells in hemato-oncological patients represent differentiated effector cells, similar to their (quantitatively minor) counterpart in healthy donors. The reason for their expansion and their pathophysiologic significance, however, remains unclear.

Influence of intrapyloric botulinum toxin injection on gastric emptying and meal‐related symptoms in gastroparesis patients

Recent observations in limited numbers of patients suggest a potential benefit of intrapyloric injection of botulinum toxin in the treatment of gastroparesis.

Desmoplastic Infantile Ganglioglioma A Potentially Malignant Tumor

Desmoplastic infantile ganglioglioma is a rare intracranial tumor of early childhood with a usually excellent prognosis despite malignant features both radiologically and histologically. We present the case of a desmoplastic infantile ganglioglioma with histologically highly anaplastic features and both intracerebral and pial metastases. After partial resection the tumor was rapidly progressive and new metastases appeared. A combination of vincristine and carboplatinum was used according to the Low Grade Glioma Protocol of the International Society of Pediatric Oncology, with a temporary good response. When histologically characterized by highly anaplastic features, it seems the biologic behavior of this tumor remains uncertain. The aggressive behavior and the responsiveness to chemotherapy in this case may challenge the belief in the benign nature of these rare tumors.

Interaction of CTLA‐4 (CD152) with CD80 or CD86 inhibits human T‐cell activation

Occupancy of CTLA‐4 (cytotoxic T‐lymphocyte antigen‐4 or CD152) negatively regulates the activation of mouse T lymphocytes, as indicated by the fate of CTLA‐4‐deficient mice, by the impact of anti‐CTLA‐4 monoclonal antibodies (mAbs) on mouse T‐cell activation in vitro and by the impact of CTLA‐4 blockade on the course of experimental tumoral, autoimmune, alloimmune or infectious disease in this animal. The function of human CTLA‐4, however, remains less clear. The expression and function of human CTLA‐4 were further explored. CTLA‐4 was expressed under mitogenic conditions only, its expression being, at least partially, dependent on the secretion of interleukin‐2. Memory T cells expressed CTLA‐4 with faster kinetics than naive T cells. The functional role of human CTLA‐4 was assessed utilizing a panel of four anti‐CTLA‐4 mAbs that blocked the interaction between CTLA‐4 and its ligands. These mAbs, in immobilized form, profoundly inhibited the activation of T cells by immobilized anti‐CD3 mAb in the absence of anti‐CD28 mAb, but co‐stimulated T‐cell activation in the presence of anti‐CD28 mAb. Finally, and importantly, blockade of the interaction of CTLA‐4 with its ligands using soluble anti‐CTLA‐4 mAbs, in intact form or as Fab fragments, enhanced T‐cell activation in several polyclonal or alloantigen‐specific CD80‐ or CD80/CD86‐dependent assays, as measured by cytokine production, cellular proliferation or cytotoxic responses. It is concluded that interaction of CTLA‐4 with its functional ligands, CD80 or CD86, can down‐regulate human T‐cell responses, probably by intracellular signalling events and independent of CD28 occupancy.

Regulation of the IL-10 Production by Human T Cells

Interleukin (IL)‐10, an immunomodulatory cytokine predominantly produced by monocytes/macrophages and T cells, inhibits several functions of dendritic cells (DC), monocytes and T cells including their cytokine production, but it stimulates B cell immunoglobulin (Ig) production and cytotoxic T lymphocyte (CTL) generation. A precise knowledge of the mechanisms that control the IL‐10 production is therefore highly important for understanding the immunoregulation. The IL‐10 production was studied in cultures of freshly isolated human T cells. A rise in intracellular calcium as well as the common γ‐chain containing cytokine receptor triggering or CD28 triggering were found to be important signals for IL‐10 induction. CD80, CD58, rIL‐12 and rIFN‐α all had efficacious and independent costimulatory activities on the IL‐10 production, while PGE2 was inhibitory. Dependence on autocrine IL‐2 signalling was shown by the effects of anti‐IL‐2 and anti‐IL‐2R monoclonal antibodies (MoAb), but the IL‐10 production proceeded partly IL‐2‐independent when CD80 provided costimulation. Sensitivity to inhibition by CsA was not removed by CD80 or CD58 costimulation and/or by addition of rIL‐12 or rIFN‐α, pointing to the absolute requirement for calcineurin activity. These data reveal important differences in the regulatory pathways between IL‐10 (a cytokine‐inhibitory interleukin) and IL‐2 (a cytokine‐inducing interleukin), which can potentially be exploited therapeutically. The fact that CsA blocks the production of IL‐10, which itself has important immunosuppressive properties, should be taken into account in defining immunosuppressive treatment schedules which include the use of CsA.

Disease- and treatment-related elevation of the neurodegenerative marker tau in children with hematological malignancies.

Children acquire neuropsychologic dysfunctions after chemotherapy for hematologic malignancy. In this study, putative changes in levels of CSF-tau (a marker of neural dysintegrity) in leukemic children prior to and during chemotherapy were studied. Cerebrospinal fluid (CSF) samples were obtained before and during treatment from patients with B cell non-Hodgkins lymphoma (NHL, n = 10), non-B cell acute lymphoblastic leukemia/NHL (non-B-ALL, n = 48), acute myeloid leukemia (AML, n = 9), other malignant diseases (n = 9), and six control children. a sandwich-type elisa (innotest htau-ag) was used for measuring csf-tau. sixteen out of 50 patients with hematological malignancies, including the patients with proven leukemic cns invasion, already showed high csf-tau levels at baseline (>300 pg/ml). The pre-induction treatment for non-B-ALL, consisting of only corticosteroids and methotrexate (MTX), resulted in a significant increase of tau at day 8 (on average to 535 pg/ml). Larger increases as compared to baseline levels of CSF-tau were observed in patients treated for B-NHL with systemic vincristine, corticosteroids and cyclophosphamide, and intrathecal MTX (mean 776 pg/ml at day 8). In two AML patients with CNS invasion, CSF-tau increased during chemotherapy up to 1500 and 948 pg/ml, respectively. In one non-B-ALL patient with MTX-induced clinical neurotoxicity, CSF-tau was above the detection limit of 2000 pg/ml. Almost one-third of the patients with hematological malignancies had elevated CSF-tau levels at diagnosis. Transient high levels of CSF-tau, reaching levels observed in other neurodegenerative disorders, were observed during induction chemotherapy for non-B-ALL, B-NHL and CNS+ AML. The clinical implications of both observations will be the subject of further study.

Inhibition of costimulation allows for repeated systemic administration of adenoviral vector in rhesus monkeys

Immunogenicity of recombinant adenoviral (Ad) vectors severely hampers the clinical development of gene therapy protocols using repeated vector administrations. Inhibition of costimulation by APCs was explored as a strategy to circumvent the immune response against Ad particles. This strategy was tested in rhesus monkeys, treated transiently with chimeric anti-human CD40 and anti-human CD86 antagonist monoclonal antibodies (MAbs) at the time of systemic administration of a recombinant Ad vector. After Ad vector administration in the absence of immunosuppressive treatment, transgene expression in the serum lasted about 3–4 weeks. All control animals developed a strong neutralizing antibody (NAb) response to the Ad particles, which totally prevented efficient administration of a second vector, as shown by the lack of transgene expression. Treatment with anti-CD40 and anti-CD86 chimeric MAbs delayed or blocked the development of a humoral response against Ad and the infiltration of CD8+ lymphocytes into the liver. This resulted in (i) increased persistence of Ad-transduced cells after injection of a first vector encoding a nonimmunogenic transgene, and (ii) the possibility of readministering a second Ad vector with significant efficacy. In both respects, the combined blockade of CD40 and CD86 was more efficient than treatment with anti-CD40 alone. This study shows for the first time in non-human primates that blocking CD40 and CD86 costimulatory molecules represents a promising strategy to inhibit immune responses against an Ad vector injected systemically.