Adenovirus-mediated CD40L gene transfer increases Teffector/Tregulatory cell ratio and upregulates death receptors in metastatic melanoma patients.

BACKGROUND AND AIMS: Malignant melanoma is an aggressive tumor sensitive for immunotherapy such as checkpoint blockade antibodies. Still, most patients with late stage disease do not respond, and the side effects can be severe. Stimulation of the CD40 pathway to initiate anti-tumor immunity is a promising alternative. Herein, we demonstrate immune profiling data from melanoma patients treated with an adenovirus-based CD40 ligand gene therapy (AdCD40L). METHODS: Peripheral blood mononuclear cells and plasma were collected from malignant melanoma patients (n = 15) enrolled in a phase I/IIa study investigating intratumoral delivery of AdCD40L with or without low dose cyclophosphamide. Cells were analyzed by flow cytometry while plasma samples were analyzed by a multi-array proteomics. RESULTS: All patients had an increased Teffector/Tregulatory cell ratio post therapy. Simultaneously, the death receptors TNFR1 and TRAIL-R2 were significantly up-regulated post treatment. Stem cell factor (SCF), E-selectin, and CD6 correlated to enhanced overall survival while a high level of granulocytic myeloid-derived suppressor cells (gMDSCs), IL8, IL10, TGFb1, CCL4, PlGF and Fl3t ligand was highest in patients with short survival. CONCLUSIONS: AdCD40L intratumoral injection induced desirable systemic immune effects that correlated to prolonged survival. Further studies using CD40 stimulation in malignant melanoma are warranted. Trial registration The 002:CD40L trial "Phase I/IIa AdCD40L Immunogene Therapy for Malignant Melanoma and Other Solid Tumors" (clinicalTrials.gov identifier: NCT01455259) was registered at September 2011.

Kick-starting the cancer-immunity cycle by targeting CD40.

Stimulation of CD40 on dendritic cells to expand and activate tumor-specific T cells and generate anticancer immunity is an attractive therapeutic approach. Since CD40 agonists exert their effects upstream of checkpoint inhibitors, including PD-1 or PD-L1 antagonists, they are ideal candidates for combination regimens.

Cytomegalovirus-specific CD4 and CD8 T cell responses in infants and children.

Congenital cytomegalovirus (CMV) infection is the most common congenital infection causing childhood morbidity. The pathogenetic mechanisms behind long-term sequelae are unclear, but long-standing viremia as a consequence of the inability to convert the virus to a latent state has been suggested to be involved. Whereas primary CMV infection in adults is typically rapidly controlled by the immune system, children have been shown to excrete virus for years. Here, we compare T cell responses in children with congenital CMV infection, children with postnatal CMV infection and adults with symptomatic primary CMV infection. The study groups included 24 children with congenital CMV infection, 19 children with postnatal CMV infection and eight adults with primary CMV infection. Among the infants with congenital CMV infection, 13 were symptomatic. T cell responses were determined by analysis of interferon gamma production after stimulation with CMV antigen. Our results show that whereas adults display high CMV-specific CD4 T cell responses in the initial phase of the infection, children younger than 2 years have low or undetectable responses that appear to increase with time. There were no differences between groups with regard to CD8 T cell function. In conclusion, inadequate CD 4 T cell function seems to be involved in the failure to get immune control of the CMV infection in children younger than 2 years of age with congenital as well as postnatal CMV infection.

An ex vivo loop system models the toxicity and efficacy of PEGylated and unmodified adenovirus serotype 5 in whole human blood.

Polyethylene glycol coating (PEGylation) of adenovirus serotype 5 (Ad5) has been shown to effectively reduce immunogenicity and increase circulation time of intravenously administered virus in mouse models. Herein, we monitored clot formation, complement activation, cytokine release and blood cell association upon addition of uncoated or PEGylated Ad5 to human whole blood. We used a novel blood loop model where human blood from healthy donors was mixed with virus and incubated in heparin-coated PVC tubing while rotating at 37 degrees C for up to 8 h. Production of the complement components C3a and C5a and the cytokines IL-8, RANTES and MCP-1 was significantly lower with 20K-PEGylated Ad5 than with uncoated Ad5. PEGylation prevented clotting and reduced Ad5 binding to blood cells in blood with low ability to neutralize Ad5. The effect was particularly pronounced in monocytes, granulocytes, B-cells and T-cells, but could also be observed in erythrocytes and platelets. In conclusion, PEGylation of Ad5 can reduce the immune response mounted in human blood, although the protective effects are rather modest in contrast to published mouse data. Our findings underline the importance of developing reliable models and we propose the use of human whole blood models in pre-clinical screening of gene therapy vectors.

Resolvin E1 reduces proinflammatory markers in human pancreatic islets in vitro.

BACKGROUND: In clinical islet transplantation, inflammatory responses initiated by the transplanted islets and by the host immune system cause acute and chronic graft loss. The resolution of acute inflammation is an active process mediated by specific signals and mediators such as resolvin E1 (RvE1). We investigated the effect of RvE1 on i) the inflammatory status of human pancreatic islets, ii) islet viability and apoptosis, and iii) the instant blood-mediated inflammatory reaction (IBMIR) IN VITRO. METHODS: Pro-inflammatory cytokines and tissue factor (TF) in isolated human islets were determined by real-time RT-qPCR (mRNA levels), CBA and Gyrolab bioaffy (protein levels) after lipopolysaccaride (LPS) stimulation. Islet viability was measured using insulin secretion in a dynamic model, ADP/ATP ratio and total ATP content. Apoptosis was measured using commercial kits after stimulation with proinflammatory cytokines. To assess effect on IBMIR, human islets were mixed with non-anticoagulated, RvE1 or vehicle pretreated ABO-compatible blood in heparin-coated tubing loops. RESULTS: Treatment of human islets with RvE1 (500 nM) for 24 h reduced LPS-induced increase in mRNA and protein levels of selected pro-inflammatory markers (IL-8, MCP-1, and TF). RvE1 lowered the ADP/ATP ratio, but had no effect on insulin secretion. RvE1 reduced the apoptotic effect of proinflammatory cytokines. Additionally, RvE1 reduced platelet consumption and TAT complex formation during the first 5 min after islet-blood contact. CONCLUSIONS: RvE1 suppresses proinflammatory markers and lowers the ADP/ATP ratio in human islets IN VITRO. RvE1 demonstrates anti-apoptotic effects in a proinflammatory milieu. Additionally, RvE1 has modest dampening effects on IBMIR. We conclude that RvE1 may have potential in clinical islet transplantation.

An oncolytic conditionally replicating adenovirus for hormone-dependent and hormone-independent prostate cancer.

The use of conditionally replicating adenoviruses offers an attractive complementary treatment strategy for localized prostate cancer. We have produced a replicating adenovirus, Ad[I/PPT-E1A], where E1A gene expression is controlled by a recombinant regulatory sequence designated PPT. The PPT sequence comprises a PSA enhancer, a PSMA enhancer and a T-cell receptor gamma-chain alternate reading frame protein promoter, and it is shielded from transcriptional interference from adenoviral backbone sequences by an H19 insulator. Ad[I/PPT-E1A] yields prostate-specific E1A protein expression, viral replication and cytolysis in vitro. Furthermore, Ad[I/PPT-E1A] considerably regresses the growth of subcutaneous LNCaP prostate cancer tumors in nude mice. Importantly, the viral replication and cytolytic effect of Ad[I/PPT-E1A] are independent of the testosterone levels in the prostate cancer cells. This may be beneficial in a clinical setting since many prostate cancer patients are treated with androgen withdrawal. In conclusion, Ad[I/PPT-E1A] may prove to be useful in the treatment of localized prostate cancer.

Identification and characterization of a novel splicing variant of vesicular monoamine transporter 1.

Vesicular monoamine transporter 1 (VMAT1) is an integral protein in the membrane of secretory vesicles of neuroendocrine and endocrine cells that allows the transport of biogenic monoamines, such as serotonin, from the cytoplasm into the secretory vesicles. The full-length VMAT1 transcript is produced from 16 exons. We have identified and characterized an alternatively spliced form of VMAT1 that lacks exon 15, the next to last exon of VMAT1. The new form was therefore denoted VMAT1Delta15. Exon 15 does not contain an even multiple of three nucleotides. As a consequence, there is a shift of reading frame, and exon 16 is translated in an alternative reading frame, yielding a novel protein with a shorter and unrelated C-terminus compared with the native VMAT1 protein. VMAT1 and VMAT1Delta15 mRNAs are simultaneously expressed in normal and neoplastic neuroendocrine cells of the GI tract. However, VMAT1 expression is always higher than VMAT1Delta15 expression. We prove that VMAT1Delta15 is not localized in large, dense core vesicles as the native form but in the endoplasmic reticulum. Furthermore, while VMAT1 can take up serotonin, VMAT1Delta15 cannot, indicating different functions for the two forms of VMAT1.

Optimization of the MB49 mouse bladder cancer model for adenoviral gene therapy.

Bladder cancer is regarded as a promising candidate for innovative therapies in the field of immune and gene therapy. In this paper, we present the subcutaneous, metastatic and a novel orthotopic model of murine MB49 bladder cancer in C57BL/6 mice. We further show the potential of using adenoviral vectors together with different transduction enhancers to augment in vivo gene delivery. Finally, we present candidate genes for tumour detection, therapy or targeting. The MB49 tumour grew rapidly in mice. The subcutaneous model allowed for tumour detection within a week and the possibility to monitor growth rate on a day-by-day basis. Injection of MB49 cells intravenously into the tail vein gave rise to lung metastases within 16 days, while instillation of tumour cells into pretreated bladders led to a survival time of 20-40 days. Adenoviral vectors can be used as a vehicle for gene transfer to the bladder. By far, the most potent transduction enhancer was Clorpactin, also known as oxychlorosene. Last, we show that MB49 cells express tumour-associated antigens like bladder cancer-4, prostate stem cell antigen and six-transmembrane epithelial antigen of the prostate. Given the possibility for efficient genetic modification of the bladder and the presence of known tumour antigens, the MB49 models can be used in innovative ways to explore immunogene therapy.

Cellular responses to cytomegalovirus in immunosuppressed patients: circulating CD8+ T cells recognizing CMVpp65 are present but display functional impairment.

The availability of tetrameric complexes of HLA class I molecules folded with immunodominant peptides makes it possible to utilize flow cytometry for rapid and highly specific visualization of virus specific CD8+ T cells. An alternate technique is to incubate whole blood with specific antigens and to subsequently detect and characterize responding T cells (e.g. by performing intracellular staining of interferon-gamma). By using an HLA-A2 tetramer construct folded with the same immunodominant CMV-peptide as that used for peptide pulsing, we monitored both the presence and functional capacity of CMV-specific CD8+ T cells. In addition T cell activation was assayed by determination of CD38 and CD69 expression. Twelve organ transplant patients and 31 healthy blood donors with latent CMV infection were investigated using CMV pp65 tetramer staining and intracellular staining of interferon-gamma after CMV pp65 peptide pulsing or CMV lysate pulsing. CMV-specific T cells were detected in similar absolute numbers as well as frequencies of T cells in the two groups investigated. However, the CMV-specific CD8+ T cells in immunosuppressed individuals showed a decreased functional response to the CMV-peptide, as evidenced by reduced interferon-gamma production when compared to healthy blood donors (19%; 42%, P < 0.005). In addition, CD38 expression was markedly higher in immunosuppressed patients compared to healthy blood donors (24%; 6%, P < 0.005). In a case report we demonstrate that reactivation of CMV can occur in an immunosuppressed patient with high number of CMV-specific T cells, but without functional capacity. Hence, these findings reflect impaired activation of cytotoxic T cells controlling latent CMV infection in immunosuppressed patients.

Human urinary bladder carcinomas express adenovirus attachment and internalization receptors.

The use of adenoviral vectors as potent gene delivery systems requires expression of the Coxsackievirus/adenovirus receptor (CVADR) on the target cell surface. This receptor is important for virus attachment to the cell surface. For effective internalization of the vector into the target cell the integrins alpha(v)beta(3) and/or alpha(v)beta(5) are needed. Since there have been reports of loss of CVADR in bladder cancer cell lines, we wanted to investigate the expression of this receptor in bladder carcinoma biopsies. Surgical biopsies, as well as five human bladder cancer cell lines, were analyzed for expression of CVADR, the integrins alpha(v)beta(3) and alpha(v)beta(5) and MHC class I. Further, we studied the ability to transduce these cell lines using adenoviral vectors. Immunohistochemistry revealed that all biopsies (27/27) were positive for CVADR. Some variation in expression was evident, and superficially growing tumors stained more strongly than invasive ones. Most human tumors expressed the integrin alpha(v)beta(5) (14/24), whereas integrin alpha(v)beta(3) was less frequently seen (3/20). The established cell lines were efficiently transduced with adenoviral vectors, and transduction could be reduced with anti-CVADR antibodies. The abundance of appropriate viral receptors on tumor biopsy cells is a further argument for using adenoviral vectors in gene therapy of bladder cancer.

Multiple myeloma cells are killed by syndecan-1-directed superantigen-activated T cells.

Multiple myeloma (MM) is an incurable plasma cell/plasmablast malignancy with a great need for innovative treatment strategies. Since experimental immunotherapy with targeted superantigens (SAg) proved to be effective in other haematopoietic tumours, we investigated whether this would also hold true for MM. We used the bacterial SAg Staphylococcus enterotoxin A (SEA), a potent activator of T cell cytotoxicity by means of its binding to particular T cell receptor Vbeta sequences on effector cells and MHC class II molecules on target cells. To eliminate potentially unspecific binding via MHC class II, SEA was point mutated (SEAm). In a second step SEAm was genetically fused to protein A (PA), resulting in a fusion protein (PA-SEAm). This fusion protein was used together with four different plasma-cell-specific/associated mAbs to direct T cells towards 10 MM target cell lines. Three of these mAbs were directed against syndecan-1/CD138, known to be highly expressed on MM and plasma cells, but absent on other haematopoietic cells. All MM cell lines proved to be sensitive to SAg-activated T cell killing (15-50% lysis), as measured in a 51Cr-release assay. This effect was clearly mediated via the plasma-cell-reactive antibodies, as control antibodies only conferred a low background lysis. MM therapy based on targeted SAgs could in theory be hampered by dysfunctional T cells in MM patients. However, we show that T cells from MM patients and healthy controls responded equally well to activation by SAg.

Potent antitumor effects of CD154 transduced tumor cells in experimental bladder cancer.

PURPOSE: Current intravesical immunotherapy for bladder cancer with bacillus Calmette-Guerin instillations is standard treatment for patients with high risk superficial tumors but relapses are common. We evaluated the tumor vaccine concept in murine bladder cancer by comparing tumor cell transduction with genes coding for the immunostimulatory molecules CD154, interleukin (IL)-12 and CD80 to design a novel vaccination strategy. MATERIALS AND METHODS: Adenoviral vectors were used to transduce murine bladder cancer MB-49 cells with genes coding for CD154, IL-12 and CD80. Parental or transduced MB-49 cells were injected subcutaneously into syngeneic mice. The effects of transgene expression on tumorigenicity and the generation of protective immunological memory against challenge with parental tumor were studied. RESULTS: All 76 animals injected with parental MB-49 cells had tumors within 8 to 12 days. Tumor cell expression of CD154 combined with IL-12 completely inhibited tumor outgrowth with all 21 mice tumor-free and CD154 transduction alone was almost as effective with 33 of 35 tumor-free. IL-12 production by tumor cells delayed tumor outgrowth and 4 of 10 mice remained tumor-free. Over expression of CD80 had no effect on tumorigenicity. CD154 expressing tumors were rapidly infiltrated with large numbers of CD4+ and CD8+ T cells. Mice vaccinated 4 times with adenoviral CD154 transduced MB-49 cells were completely protected against challenge with parental tumor. Co-injection of CD154 modified cells with parental MB-49 cells retarded tumor growth. CONCLUSIONS: Our experimental results suggest that the potent antitumor effects of CD154 gene transduction should be considered for immunostimulatory gene therapy for bladder cancer.

Circulating soluble vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in immunocompetent and renal transplant patients: correlation with cytomegalovirus disease and renal function.

The plasma levels of the soluble adhesion molecules, soluble vascular cell adhesion molecule-1 (sVCAM-1) and intercellular adhesion molecule-1 (sICAM-1), were measured before and after transplantation in 26 renal transplant recipients, and in 173 longitudinally collected samples in 17 of the patients. The patients were carefully monitored for the presence of cytomegalovirus (CMV) infection and rejection. Forty healthy blood donors and 12 otherwise healthy subjects with symptomatic primary CMV infections served as controls. During CMV disease, plasma levels of sVCAM-1 and sICAM-1 were elevated in both renal transplant patients and otherwise healthy subjects with CMV disease. The sVCAM-1 levels were strongly elevated before transplantation in renal transplant recipients and correlated with creatinine levels. Increased sVCAM-1 levels were also registered during rejection episodes. CMV disease, per se, is associated with markedly increased levels of sVCAM-1 and sICAM-1. There is also a correlation of sVCAM-1 levels with serum creatinine levels. Thus, the presence of CMV infection and renal function are factors that must be considered in further studies of soluble adhesion molecules.

Characterization of CMVpp65-specific CD8+ T lymphocytes using MHC tetramers in kidney transplant patients and healthy participants.

BACKGROUND: Cytomegalovirus (CMV) is a ubiquitous herpesvirus that infects 50-90% of individuals in different populations. After primary infection, the virus persists latently in myeloid cells under the control of specific T-cells. Reactivation of CMV infection may cause lethal organ dysfunction and is frequently seen in immunosuppressed individuals. CD8+ cytotoxic T-cells (CTL) have a primary role in suppressing CMV reactivation, and the dominating CTL response is directed against pp65. METHODS: MHC tetramers, that is, complexes between HLA class I (or class II) molecules and antigenic peptides conjugated to fluorochromes allow the direct visualization of antigen-specific receptor-carrying T-cells using flow cytometry. We constructed a novel MHC tetramer for identification of CMVpp65-specific CD8+ T-cells using HLA-A2 molecules folded with the immunodominant NLVPMVATV peptide. RESULTS: The A2/pp65 tetramer specifically stained CMV-directed T-cell lines, and sorted cells showed CMV-specific cytotoxicity. High proportions (0.1-9%) of the CD8+ T-cells were A2/pp65 tetramer+ in healthy HLA-A2+ CMV carriers and in immunosuppressed kidney transplant patients with latent infection. Patients with reactivated CMV infection exhibited up to 15% A2/pp65 tetramer+ cells, which seemed to correlate with CMV load over time. A2/pp65 tetramer+ cells expressed T-cell activation markers. CONCLUSIONS: The construction of a novel A2/pp65 MHC tetramer enabled the design of a rapid and precise flow cytometric method allowing quantitative and qualitative analysis of CMV-specific T-cells. The number of A2/pp65 tetramer binding CTLs in blood may prove to be clinically relevant in assessing the immune response to CMV.

Staphylococcus aureus Cowan strain 1 activation of B-chronic lymphocytic leukaemia cells augments the response to CD40 stimulation.

The signals involved in regulating the proliferation, differentiation and survival of B-chronic lymphocytic leukemia (B-CLL) cells are fully understood. B-CLL cells have been found to respond poorly to various activation signals and only after successful Epstein-Barr virus (EBV) transformation has it been possible to maintain such cells in long-term cultures. In this work we describe a new method to activate and induce proliferation in B-CLL cells and to maintain such cells in long-term culture for longer than 1 month. We used a combination of protocols in an attempt to mimic some of the signals of a thymus-dependent immune response. The B-CLL cells were first activated with Staphylococcus aureus Cowan strain 1 (SAC) particles plus thioredoxin (Trx), followed by stimulation with interleukin (IL)-2 + Trx. This treatment primed the cells for further stimulation with anti-CD40 monoclonal antibody (MoAb) presented on irradiated CD32L cells (the CD40-system) or soluble CD40 Ligand, and a combination of Trx and cytokines (IL-4 + IL-10), which allowed the cells to be maintained for up to 1 month with preserved viability and a variable rate of proliferation. However, induced proliferation of the B-CLL cells was limited to approximately 1 month, suggesting that additional signals are required to facilitate further proliferation.

Serum levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) are elevated in advanced stages of non-Hodgkin's lymphomas.

The serum levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) were measured in 116 patients with non-Hodgkin's lymphomas (NHL) tested previously for soluble intercellular adhesion molecule-1 (sICAM-1). In contrast to Hodgkin's disease and chronic lymphocytic leukaemia, the sVCAM-1 levels in NHL patients were not significantly different from the levels of healthy controls (n = 31). However, sVCAM-1 was elevated in advanced stage disease, i.e. stages III + IV. Elevated serum levels of sVCAM-1 were associated with significantly poorer disease-free (p = 0.024) and overall (p = 0.02) survival. sVCAM-1 correlated poorly with other known prognostic variables (LDH, sTK and beta 2m) and with sICAM-1. None of the tested markers added prognostic information for disease-free survival independently of Ann Arbor stage and B-symptoms. The expression of VCAM-1 and ICAM-1 in tumour biopsies from 15 patients representing 7 different histologies were examined and compared with the serum levels of the soluble adhesion molecules. No correlation was found between the adhesion molecule expression by vascular endothelium and the corresponding serum levels.

Serum levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) are elevated in advanced stages of non-Hodgkin's lymphomas.

The serum levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) were measured in 116 patients with non-Hodgkin's lymphomas (NHL) tested previously for soluble intercellular adhesion molecule-1 (sICAM-1). In contrast to Hodgkin's disease and chronic lymphocytic leukaemia, the sVCAM-1 levels in NHL patients were not significantly different from the levels of healthy controls (n=31). However, sVCAM-1 was elevated in advanced stage disease, i.e. stages III+IV. Elevated serum levels of sVCAM-1 were associated with significantly poorer disease-free (p = 0.024) and overall (p = 0.02) survival. sVCAM-1 correlated poorly with other known prognostic variables (LDH, sTK and beta2m) and with sICAM-1. None of the tested markers added prognostic information for disease-free survival independently of Ann Arbor stage and B-symptoms. The expression of VCAM-1 and ICAM-1 in tumour biopsies from 15 patients representing 7 different histologies were examined and compared with the serum levels of the soluble adhesion molecules. No correlation was found between the adhesion molecule expression by vascular endothelium and the corresponding serum levels.

Targeted superantigens for immunotherapy of haematopoietic tumours.

With the exception of childhood common acute lymphoblastic leukaemia (cALL), treatment of other hematopoietic B cell lineage tumours such as non-Hodgkin's lymphoma (B-NHL), adult ALL and multiple myeloma (MM) is unsatisfactory. Similarly, the therapeutic outcome of acute and chronic myeloid leukaemia (AML, CML) is frequently dismal. At the same time, leukaemia/lymphoma cells represent ideal targets for immunotherapy. The present review summarizes our preclinical experience with a novel type of cytotoxic T cell based immunotherapy for B-lineage and myeloid tumours. Staphylococcal enterotoxin-derived superantigens (SAgs) are among the most potent T cell activators known, linking the T cell receptor to HLA-DR on natural target cells. SAgs were genetically engineered to reduce DR binding and were then fused to Fab parts of tumour-directed monoclonal antibodies (mAbs). Using these "targeted" SAgs, highly efficient lysis of B-lineage (B-NHL, B-CLL, ALL, MM) and myeloid (AML, CML) tumour cells by T-cells was achieved in vitro and in an animal model. We are entering an interesting era of innovative cancer therapy based on novel man-made biotherapeutic agents.

Soluble vascular cell adhesion molecule-1 (sVCAM-1) is an independent prognostic marker in Hodgkin's disease.

Serum levels of soluble vascular cell adhesion molecule-1 (sVCAM-1. sCD106) were significantly elevated in patients with Hodgkin's disease (HD) (n = 101) compared to controls (n= 31) (P<0.0001). sVCAM-1 correlated with histology, stage, B-symptoms, and prognostic markers (sICAM-1, sCD30, sIL-2R, LDH). sVCAM-1, sICAM-1 and sCD30 added independent prognostic information for both disease-free and overall survival. 14 biopsies from 13 patients with HD were immunostained for VCAM-1 and ICAM-1. The vascular endothelium stained positive for VCAM-1 in 10/12 evaluable biopsies and for ICAM-1 in all evaluable biopsies. A stromal expression of both adhesion molecules precluded a precise evaluation of HRS-cells. This led us to investigate VCAM-1 (and ICAM-1) expression in six Hodgkin cell lines (HDLM-2, L428, L540, L591, DEV, KM-H2). Two cell lines stained positive for VCAM-1 (HDLM-2, L591). All cell lines stained positive for ICAM-1. sVCAM-1 is a new prognostic marker in HD; its predictive power equals or surpasses that of sCD30 and sICAM-1. Furthermore, two Hodgkin cell lines stained positive for VCAM-1. This indicates that VCAM-1 may be expressed by some HD tumour cells in vivo.

Antibody-directed superantigen-mediated T-cell killing of myeloid leukaemic cell line cells.

Bacterial superantigens (SAgs) bound to MHC class II molecules on target cells are efficient activators of cytotoxic T cells expressing certain T cell receptor (TCR) Vbeta regions We described earlier that the specificity of the SAg Staphylococcus enterotoxin A (SEA) can be changed by introducing a D227A point mutation in the major MHC class II binding site and by genetically fusing the SEA mutant (SEAm) to protein A (PA). This SEAm-PA fusion protein can then be used to direct cytotoxic T cells to tumour cells coated with monoclonal antibodies (mAbs). In this communication, we tested the PA-SEAm fusion protein together with mAbs against the myeloid cell surface antigens CD13, CD15 and CD33. A SEA-reactive T cell line was used as effector cells against 10 different myeloid leukaemic cell lines. Optimal lysis of antigen positive leukaemic cells was obtained at a PA-SEAm concentration of 1 ng/ml and effector : target cell ratios of 15 : 1. No correlation between target cell sensitivity and the level of surface antigen expression could be seen. The 6 acute myeloid leukaemia (AML) cell lines tested appeared to be more sensitive than the 4 chronic myeloid leukaemia (CML) cell lines. The sensitivity of the AML cell line HL-60 could be improved further by stimulation with TNFalpha. This was accompanied by increased surface ICAM-1 expression whereas specific target molecule expression (CD13, CD33) was unchanged. This suggests that sensitivity to lysis is related to the leukaemic subtype and ICAM-1 expression but not to the tumour antigen density. Our results show that it is possible to direct cytotoxic T cells to myeloid leukaemia cells by using SAgs linked to mAbs, and encourage the construction and testing of a recombinant direct SAg-mAb fusion protein as a candidate drug for therapy of myeloid leukaemias.