Wilms' tumor gene 1 immunotherapy in pelvic gynecological malignancies.

Pelvic gynecological malignancies account for 6% of all cancers. In the relapsed state, classical treatments are limited. There is an urgent need for new and personalized treatment. Wilms' tumor gene 1 (WT1) is the most important tumor-associated antigen. Although highly present in gynecological tumors, active immunotherapy against it is still underexplored. This review gives an insight into the importance of WT1 in pelvic gynecological malignancies and the first taken steps into the world of WT1 immunotherapy.

In vivo bioluminescence imaging in an experimental mouse model for dendritic cell based immunotherapy against malignant glioma.

The value of bioluminescence imaging (BLI) for experimental cancer models has become firmly established. We applied BLI to the GL261 glioma model in the context of dendritic cell (DC) immunotherapy. Initial validation revealed robust linear correlations between in vivo, ex vivo and in vitro luciferase activity measurements. Ex vivo BLI demonstrated midline crossing and leakage of tumor cells. Orthotopically challenged mice followed with BLI showed an initial adaptation phase, after which imaging data correlated linearly with stereologically determined tumor dimensions. Transition from healthy to moribund state corresponded with an increasing in vivo flux but the onset of neurological deficit was clearly delayed compared to the onset of in vivo flux increase. BLI was implemented in prophylactic immunotherapy and imaging data were prognostic for therapy outcome. Three distinct response patterns were detected. Our data underscore the feasibility of in vivo BLI in an experimental immunotherapeutic setting in the GL261 glioma model.

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.

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.

Blocking B7 and CD40 co-stimulatory molecules decreases antiviral T cell activity.

Inhibition of co-stimulatory signals for T cells by interrupting CD80/CD86-CD28 and CD40-CD154 interactions is a promising approach to prevent transplant rejection and to induce graft tolerance. However, this tolerizing treatment might affect T cell reactivity towards all the antigens to which the immune system is exposed during treatment. We addressed the question whether such inhibition of co-stimulatory ligands on human antigen presenting cells (APC) would affect T cell reactivity against a virus. This was tested in an in vitro system with freshly isolated human monocytes transduced with adenovirus (ad) containing either murine interferon-gamma (mIFN-gamma) or green fluorescent protein (GFP) as marker transgene. T cells co-cultured with transduced monocytes proliferated and produced cytokines. These 'primed' T cells had strong antiviral activity as they subsequently killed ad/GFP-transduced monocytes and reduced mIFN-gamma accumulation in coculture with ad/mIFN-transduced monocytes. However, if priming had occurred in the presence of blocking anti-CD40/CD80/CD86 MoAbs, generation of this antiviral activity was completely prevented. Moreover, T cells primed in the absence of co-stimulatory cells failed to proliferate upon restimulation with adenovirus-transduced monocytes. The results confirm that co-stimulatory signals from APC are required for efficient induction of antiviral T cell activity and point to a potential infectious risk of blocking co-stimulatory signals.

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 gamma-chain containing cytokine receptor triggering or CD28 triggering were found to be important signals for IL-10 induction. CD80, CD58, rIL-12 and rIFN-alpha 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-alpha, 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.

Effects of co-stimulation by CD58 on human T cell cytokine production: a selective cytokine pattern with induction of high IL-10 production.

CD58 is the ligand for the CD2 molecule on human T cells and has been shown to provide a co-stimulatory signal for T cell activation. However, its physiological role is still unclear. We studied the effects of co-stimulation by CD58 on the production of T(h)1-type (IL-2- and IFN-gamma) or T(h)2 type (IL-4, IL-5 and IL-10) cytokines in an in vitro culture system of purified human T cells with CD58-transfected P815 cells and with anti-CD3 as the primary stimulus. Co-stimulation of T cells by CD58 potently induced IL-10 and IFN-gamma production (at the protein and at the mRNA level), and transforming growth factor-ss production (at the mRNA level), comparable to what can be found in CD80 co-stimulated T cell cultures. In contrast, we found low to absent IL-2, IL-4, IL-5, IL-13 and tumor necrosis factor-alpha production after CD58 co-stimulation, and this was not due to suppressive effects of endogenously produced IL-10. CD80 co-stimulation strongly induced all these cytokines. Intracellular staining for cytokine expression revealed the existence of a T cell subpopulation induced by CD58 co-stimulation to produce both IFN-gamma and IL-10. We furthermore found that the selective cytokine profile induced by CD58 co-stimulation is further accentuated by rIL-12 and by rIFN-alpha. Using cyclosporin A as an inhibitor of the calcineurin enzyme, we could show that production of all cytokines in this system is calcium dependent. CD58 co-stimulation thus induces a cytokine pattern corresponding to that described for T regulatory (T(r)) 1 cells and to the pattern reported to be induced by the newly identified B7 family member, B7-H1.

Differences in regulatory pathways identify subgroups of T cell-derived Th2 cytokines.

We analysed regulatory mechanisms involved in the production of Th2 cytokines by freshly isolated human T cells. We used an in vitro culture system in which the primary signal was provided by a cross-linking anti-CD3 MoAb presented on the Fc receptors of P815 cells. Both CD80 and CD86, expressed on transfected P815 cells, were able to provide efficient costimulation for the production of IL-4, IL-5 and IL-13. IL-2 was also highly important for induction of all three Th2 cytokines. However, differences between IL-4 on the one hand and IL-5 and IL-13 on the other hand were observed when sensitivity to cyclosporin A (CsA) was studied. CsA (an inhibitor of calcineurin phosphatase activity) strongly inhibited IL-4 production, but it did either not affect or even increased IL-5 and IL-13 production. In accordance with this, CD80 and phorbol myristate acetate (PMA) (without anti-CD3 or calcium ionophore) were sufficient to induce production of IL-5 and IL-13, but not of IL-4. The subgrouping of Th2 cytokines was further confirmed at another level on the basis of differences in cell sources: IL-4 was predominantly produced by CD4+ T cells, while IL-5 and IL-13 were produced by both CD4+ and CD8+ T cells. Thus, differences in cell sources and in the requirement of the calcium/calcineurin-signalling pathway allowed us to identify two subgroups (IL-4 and IL-5/IL-13) among human Th2-type T cell cytokines.

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-Hodgkin's 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 1,500 and 948 pg/ml, respectively. In one non-B-ALL patient with MTX-induced clinical neurotoxicity, CSF-tau was above the detection limit of 2,000 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.

Naive human T cells can be a source of IL-4 during primary immune responses.

IL-4 plays a key role in driving the differentiation of CD4+ Th precursors into Th2 cells, both in mice and in humans. The source of IL-4 during primary immune responses is, however, still debated. When IL-4 consumption in in vitro T cell cultures was blocked with a MoAb to the IL-4 receptor alpha-chain (IL-4Ralpha), it became evident that freshly isolated naive (CD45RO-) CD4+ T cells from adults or cord blood produce IL-4 upon activation with anti-CD3 and CD80. IL-4 production by naive T cells is strictly IL-2-dependent. Endogenous IL-4 activity in naive CD4+ T cell cultures modulates the production of interferon-gamma (IFN-gamma) on the one hand and IL-5 and IL-13 on the other hand in opposite directions, and it is partly responsible for the low IFN-gamma production by cord blood T cells. Comparison of the ratio of IL-4/IFN-gamma in supernatants of T cell cultures reveals a skewing towards IL-4 production by cord blood T cells, while naive T cells from (non-atopic) adults predominantly produce IFN-gamma. We conclude that CD4+ naive T cells can produce IL-4 without the need for Th2 differentiation, and therefore that they can be the initial source of IL-4 required at the time of priming for T cell differentiation into Th2 cells.

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.

Blocking CD40 - CD154 and CD80/CD86 - CD28 interactions during primary allogeneic stimulation results in T cell anergy and high IL-10 production.

Although CD28 triggering provides an important co-stimulatory signal to T cells, blocking the CD80/CD86 - CD28 interaction with CTLA-4lg fusion protein is not sufficient for tolerance induction in vivo or in vitro. According to more recent data, interruption of the CD40 - CD154 interaction might complement the effect of CTLA-4lg and induce graft acceptance. We studied the effects of a blocking anti-CD40 monoclonal antibody (mAb) and/or blocking anti-CD80/anti-CD86 mAb in cultures of human peripheral blood mononuclear cells (PBMC) stimulated with allogeneic PBMC. T cells activated by alloantigens in the presence of anti-CD80, anti-CD86 and anti-CD40 entered a state of alloantigen-specific non-responsiveness as evidenced upon restimulation by lack of proliferation, cytotoxic activity, and IL-2, IL-5 and IL-13 production. IFN-gamma production during restimulation was less than in the control cultures, while the production of IL-10 was enhanced. Addition of recombinant IL-2 during the restimulation rescued alloantigen-specific activity. We conclude that the simultaneous blocking of the CD40 - CD154 and CD80/CD86 - CD28 interaction during allogeneic T cell activation induces T cell anergy. Since anergic cells induced by this treatment still produce high levels of IL-10, the latter could contribute to modulation of antigen-presenting cell activity and to bystander suppression of residual reactive T cells.

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-gamma, and TNF-alpha 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.

Peripheral blood lymphocyte subset shifts in patients with untreated hematological tumors: evidence for systemic activation of the T cell compartment.

Flow cytometry immunophenotyping of peripheral blood lymphocyte subsets and multivariate data-analytical techniques revealed that among untreated hemato-oncological patients (n = 48) with lymphomas, acute and chronic myeloid and lymphocytic leukemias, monoclonal gammopathy of undetermined significance, and multiple myeloma, 42% had (nonmalignant) lymphocyte profiles clearly distinct from healthy donors. Notably, a similar pattern of increased CD3+ CD57+, CD3+ HLA-DR+, CD3+ CD(16 + 56)+, CD4- CD8+, CD8+ CD57+, CD8+ CD28-, and CD8+ CD62L- subsets was detected. More extensive three-color immunophenotyping on an additional group of 49 untreated patients revealed that both CD4+ and CD8+ T cells displayed significant increases of activation markers: CD69, CD(16 + 56), HLA-DR, CD71, and CD57, and a loss of CD62L and CD28, which is also interpreted as a sign of activation. Consistent with the phenotypical signs of in vivo immune activation, polyclonal cytolytic activity, measured ex vivo in an anti-CD3-redirected assay, was detected within immunomagnetically purified CD4+ T cells of three out of six B-CLL patients investigated, but not within purified CD4+ T cells of five healthy donors. The purified CD8+ T cells of patients (n = 28) and donors (n = 5) on the other hand displayed similar polyclonal cytotoxic activities at the various effector:target ratios investigated. Tumor-directed cytotoxic activity of purified CD4+ (n = 6) and/or CD8+ T cells (n = 15) against freshly isolated autologous tumor cells was not detected in any of the experiments. Collectively, our results demonstrate systemic T cell activation as a common feature in hematological neoplasia, and a markedly enhanced cytolytic activity of the CD4- subset in CLL patients. The reason(s) for this expansion of activated T cells and its pathophysiologic significance, however, remain unclear.

Cyclosporin A increases IFN-gamma production by T cells when co-stimulated through CD28.

Despite its calcineurin-inhibiting properties, cyclosporin A (CsA) can not inhibit IL-2 production when T cells are co-stimulated by CD80/CD86 on the antigen-presenting cells. We studied the in vitro effect of CsA on IFN-gamma production. Anti-CD3 monoclonal antibody (mAb) was used as the primary stimulus for activation of purified human T cells. A stimulating anti-CD28 mAb, or CD80 or CD86 on stably transfected P815 cells, provided the co-stimulatory signal. IL-2 production was hardly affected by CsA under these stimulating conditions, while IFN-gamma (at the protein and mRNA level) was markedly stimulated by CsA. The use of anti-CD3 or phorbol 12-myristate 13-acetate with ionomycin as the primary stimulus, together with costimulation through either CD28 or CD2 using transfectants with the appropriate ligands, allowed us to demonstrate that the resistance of IFN-gamma production to inhibition by CsA required both CD3 and CD28 triggering. Inhibition of IL-10 production, and to a lesser degree of IL-4 production, by CD4+ cells was responsible for the enhancement of IFN-gamma production in the presence of CsA. In conclusion, IFN-gamma production by CD28-co-stimulated CD4+ T cells is resistant to inhibition by CsA and can even be facilitated by CsA as a result of removing a negative regulatory signal which is mainly IL-10 mediated. This finding might have implications for immunosuppressive strategies based upon the use of CsA.

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-alpha) and interferon-gamma (IFN-gamma)) 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.