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Biomedical subjects

M Dohlsten

Publications and source records attributed to M Dohlsten.

At least 19 recordsLinked to original sources
PubMed2001

Therapy of human non-small-cell lung carcinoma using antibody targeting of a modified superantigen.

Superantigens activate T-cells by linking the T-cell receptor to MHC class II on antigen-presenting cells, and novel reactivity can be introduced by fusing the superantigen to a targeting molecule. Thus, an antibody-targeted superantigen, which activates T cells to destroy tumour cells, might be used as cancer therapy. A suitable target is the 5T4 oncofetal antigen, which is expressed on many carcinomas. We constructed a fusion protein from a Fab of a monoclonal antibody recognizing the 5T4 antigen, and an engineered superantigen. The recombinant product 5T4FabV13-SEA(D227A)bound the 5T4 antigen expressed on the human non-small-cell lung cancer cell line Calu-1 with a Kd of 1.2 nM while the substitution of Asp227 to Ala in the superantigen moiety reduced binding activity to MHC class II. 5T4FabV13-SEA(D227A)tumour reactivity was demonstrated in 7/7 NSCLC samples by immunohistochemistry, while normal tissue reactivity was low to moderate. 5T4FabV13-SEA(D227A)induced significant T-cell-dependent in vitro killing of sensitive 5T4 bearing Calu-1 cells, with maximum lysis at 10(-10)M, while the capacity to lyse MHC class II expressing cells was approximately 1000 times less effective. Immunotherapy of 5T4FabV13-SEA(D227A)against human NSCLC was investigated in SCID mice reconstituted with human peripheral blood mononuclear cells. Mice carrying intreperitoneally growing Calu-1 cells showed significant reduction in tumour mass and number after intravenous therapy with 5T4FabV13-SEA(D227A). Thus, 5T4FabV13-SEA(D227A)has highly attractive properties for therapy of human NSCLC.

Animals↗
PubMed2001

Identification of a CD28 response element in the CD40 ligand promoter.

Ligation of the T cell coreceptor CD28 or CD2 by its cognate ligands B7-1 or LFA-3, respectively, greatly aids the Ag-induced up-regulation of several genes, including IL-2 and CD40 ligand (CD40L). Using luciferase reporter constructs under the control of the 1.2 kb of 5' noncoding region of the human CD40L gene, we have found that stimulation through CD28 was required for a strong transcriptional activity of the CD40L promoter in response to TCR ligation, while the activity induced by CD2 was slightly lower than CD28. Deletion analysis demonstrated that the transcriptional elements mediating this effect were located within a 300-bp region upstream of the start site. Further dissection of this region and gel shift analyses demonstrated the presence of a CD28 response element in a region located between nucleotides -170 to -164 relative to the start site. Transcriptional studies with a CD40L enhancer-promoter carrying a mutation in this putative CD28 response element revealed that the activity was reduced by 80 and 70% after B7-1 and LFA-3 costimulation, respectively. The transcription factor complex bound to this site contained at least JunD, c-Fos, p50, p65, and c-REL:, but not c-Jun. Mutations introduced into the CD28RE also blocked the binding of this complex. These observations identify an important role for the CD28 signaling pathway in the regulation of CD40L promoter transcriptional activity.

Animals↗
PubMed2001

Antagonistic effects of the staphylococcal enterotoxin a mutant, SEA(F47A/D227A), on psoriasis in the SCID-hu xenogeneic transplantation model.

Psoriasis is a T-cell-mediated immune dermatosis probably triggered by bacterial superantigens. This pathomechanism has been experimentally reproduced in a SCID-hu xenogeneic transplantation model. We analyzed the effects of different bacterial superantigens on the induction of psoriasis in this model. Staphylococcal enterotoxin B and exfoliative toxin triggered the onset of psoriasis when administered repetitively intracutaneously over a period of 2 wk, whereas staphylococcal enterotoxin A representing a distinct subfamily of staphylococcal enterotoxins only mimicked certain aspects of psoriasis. The biologic effects of staphylococcal enterotoxin A were more pronounced when a mutated form, SEA(H187A), of this superantigen with reduced affinity to major histocompatibility complex class II was coinjected. Another mutated variant, SEA(F47A/D227A), exhibiting no measurable major histocompatibility complex class II affinity blocked the effects triggered by wild-type staphylococcal enterotoxin A when injected in a 10-fold higher dose. Inhibition was specific as induction of psoriasiform epidermal changes by staphylococcal enterotoxin B could not be blocked. As staphylococcal enterotoxin A, in contrast to the other superantigens tested, is capable of inducing epidermal thickening but not the typical appearance of psoriasis, we conclude that bacterial superantigens may differ with regard to their effects on human nonlesional psoriatic skin. Staphylococcal-enterotoxin-A-mediated effects were blocked by a genetically engineered superantigen highlighting the potential therapeutic use of mutated superantigens.

Animals↗
PubMed2000

IL-2 unresponsiveness in anergic CD4+ T cells is due to defective signaling through the common gamma-chain of the IL-2 receptor.

Repeated administration of the superantigen staphylococcal enterotoxin A to mice transduces a state of anergy in the CD4+ T cell compartment, characterized by inhibition of IL-2 production and clonal expansion in vivo. In contrast to what has been reported on anergic T cell clones in vitro, culture of in vivo anergized CD4+ T cells in the presence of exogenous IL-2 did not overcome the block in responsiveness. In this study, we demonstrate that CD4+ T cells from mice anergized with staphylococcal enterotoxin A also exhibit a reduced proliferative capacity in response to IL-7 and IL-15, cytokines that share a common gamma-chain with the IL-2R. Flow-cytometric analysis revealed only modest changes in the expression of the different IL-2R chains. In a number of experiments, our results also provide evidence that excludes a major role of the IL-2R alpha-chain in this system. According to these results, the inability of anergic cells to respond to IL-2 is not mainly due to a down-regulation of the high affinity IL-2R, but to a perturbation in intracellular signaling. Our study confirmed that the activation and tyrosine phosphorylation of Janus-associated kinase 3 and STAT5 were considerably weaker after anergy induction. Moreover, anergic CD4+ T cells showed significantly reduced DNA-binding ability to STAT5-specific elements. Taken together, we suggest that the observed IL-2 unresponsiveness in anergic CD4+ T cells could be due to a defect in signaling through the common gamma-chain of the IL-2R.

Animals↗
PubMed2000

Vaccination with B16 melanoma cells expressing a secreted form of interleukin-1beta induces tumor growth inhibition and an enhanced immunity against the wild-type B16 tumor.

We have demonstrated previously that gene transfer of the mature human interleukin-1beta (IL-1beta) gene, fused to a signal sequence (ss), into mouse B16 melanoma cells results in an inhibition of their growth in vivo compared with control B16 cells. We here extend these results to show that intraperitoneal vaccinations with irradiated IL-1beta-secreting cells result in protection against subsequent subcutaneous challenge with wild-type (wt) B16 tumor cells in syngeneic C57BL/6 mice. This protection appears to be long-lasting, because rechallenge of cured mice 4 months after the first challenge also demonstrated resistance. In addition, we demonstrate that mice with established wt tumors subjected to therapeutic vaccinations with irradiated B16/ssIL-1beta cells starting 3 days after challenge isografting have a significantly inhibited tumor growth and 25-40% survival at the challenge doses given. In vitro coculture of spleen cells from B16/ssIL-1beta vaccinated animals and wt B16 cells induced an enhanced proliferative response, which correlated with elevated production of IL-2 and interferon-gamma. A significantly enhanced cytolytic activity against B16 wt target tumor cells was observed when spleen cells from B16/ssIL-1beta vaccinated mice were used as effector cells compared with spleen cells from control vaccinated mice. In vitro depletion experiments using anti-asialo GM1 revealed a prominent role for natural killer cells as effector cells. The data suggest that local IL-1beta secretion during the vaccination phase can provoke or augment protective immune responses to B16 melanoma cells, which are otherwise not recorded in mice bearing B16 tumors.

Animals↗
PubMed2000

Selective induction of p38 mitogen-activated protein kinase activity following A6H co-stimulation in primary human CD4(+) T cells.

We have recently described the novel A6H antigen expressed on human peripheral blood T cells and on renal cell carcinoma cells. Cross-linking of the A6H antigen results in co-stimulation of human CD4(+) T cells, characterized by induction of the transcription factor activator protein-1 (AP-1), proliferation and prominent IFN-gamma production, but low levels of IL-2. The proximal signaling events associated with A6H ligation include protein tyrosine kinase phosphorylation and association of p56 Lck, ZAP-70 and the TCR zeta chain. In this study we show that A6H co-stimulation selectively induced activation of the p38 mitogen-activated protein kinase (MAPK) pathway, whereas no significant c-Jun N-terminal kinases (JNK) activity was observed. In contrast, CD28 co-stimulation resulted in both p38 and JNK MAPK activities. Human CD4(+) T cells co-stimulated with A6H up-regulated AP-1 binding proteins reactive with a proximal AP-1 binding site in the human IFN-gamma promoter and a consensus AP-1 binding site. Moreover, preincubation of the T cells with the specific p38 MAPK inhibitor SB203580 resulted in decreased AP-1 binding following A6H or CD28 co-stimulation. This suggests that the p38 MAPK pathway is required for induction of full AP-1 binding activity in human CD4(+) T cells co-stimulated with A6H or CD28. Blocking the p38 MAPK pathway by SB203580 completely inhibited IFN-gamma production from A6H co-stimulated T cells and radically reduced IFN-gamma production from T cells co-stimulated with anti-CD28. In contrast, no significant inhibition of IL-2 production was seen after blocking of the p38 MAPK in either A6H or CD28 co-stimulated T cells. Since the p38 MAPK recently has been shown to be critically involved in regulation of IFN-gamma production from T(h)1 cells, we propose that A6H co-stimulation induces a specific pathway, mediated via p38 and AP-1 activation, for induction of a T(h)1 profile in human CD4(+) T cells.

Antigens, Neoplasm↗
PubMed1999

Staphylococcal enterotoxin H displays unique MHC class II-binding properties.

Staphylococcal enterotoxin H (SEH) has been described as a superantigen by sequence homology with the SEA subfamily and briefly characterized for its in vivo activity. In this study, we demonstrate that SEH is a potent T cell mitogen and inducer of T cell cytotoxicity that possesses unique MHC class II-binding properties. The apparent affinity of SEH for MHC class II molecules is the highest affinity ever measured for a staphylococcal enterotoxin (Bmax1/2 approximately 0.5 nM for MHC class II expressed on Raji cells). An excess of SEA or SEAF47A, which has reduced binding to the MHC class II alpha-chain, is able to compete for binding of SEH to MHC class II, indicating an overlap in the binding sites at the MHC class II beta-chain. The binding of SEH to MHC class II is like SEA, SED, and SEE dependent on the presence of zinc ions. However, SEH, in contrast to SEA, binds to the alanine-substituted DR1 molecule, betaH81A, believed to have impaired zinc-bridging capacity. Furthermore, alanine substitution of residues D167, D203, and D208 in SEH decreases the affinity for MHC class II as well as its in vitro potency. Together, this indicates an MHC class II binding site on SEH with a different topology as compared with SEA. These unique binding properties will be beneficial for SEH to overcome MHC class II isotype variability and polymorphism as well as to allow an effective presentation on APCs also at low MHC class II surface expression.

Alanine↗
PubMed1999

CTLA-4 ligation suppresses CD28-induced NF-kappaB and AP-1 activity in mouse T cell blasts.

The effects of cytotoxic lymphocyte antigen 4 (CTLA-4) on CD3/CD28 monoclonal antibody (mAb) activation of CD4(+)/CTLA-4(+) blastoid T cells were studied in an in vitro model system. As previously reported, coligation of CTLA-4 mAb results in suppression of T cell proliferation and cytokine production. The proliferation but not the interleukin 2 (IL-2) production could be restored by addition of exogenous IL-2, suggesting that the inhibitory effect occurred at the level of IL-2 production rather than at the regulation of the IL-2 receptor pathway. To study the effects on nuclear factors critical for T cell activation, we analyzed the levels of the transcription factors NF-kappaB and AP-1. These were potently induced in CD3/CD28 mAb-restimulated T cells. In contrast, CTLA-4 ligation strongly suppressed the induction of both transcription factors. The compositions of NF-kappaB and AP-1 family members were similar, irrespective of stimulation conditions. Analyses of the NF-kappaB regulator IkappaB-alpha revealed similar levels of IkappaB-alpha protein in the preparations. However, a reduced phosphorylation of IkappaB-alpha in CTLA-4 coengaged T cell blasts compared with T cells ligated with CD3/CD28 was found. Previous studies have concluded that CTLA-4 ligation regulates T cell activation by inhibiting the T cell receptor-mediated signals. However, the present findings propose that the major impact of CTLA-4 ligation is inhibition of signals mediated by CD28.

Abatacept↗
PubMed1999

Long-term survival and complete cures of B16 melanoma-carrying animals after therapy with tumor-targeted IL-2 and SEA.

The bacterial superantigen (SAg) staphylococcal enterotoxin A (SEA) is a potent inducer of CTL activity and cytokine production in vivo. To engineer SAg for cancer immunotherapy, we genetically fused SEA to a Fab fragment of the C215 tumor-reactive antibody. Strong reduction of lung metastasis was seen in mice carrying established lung metastases of the poorly immunogenic B16-C215 melanoma after Fab-SEA therapy. However, important anti-tumor effector functions, such as IFN-gamma secretion and CTL activity, gradually declined during therapy. In this study, we show that Fab-SEA immunotherapy is strongly potentiated by Fab-IL-2 co-administration. Combined Fab-IL-2 and Fab-SEA therapy prolongs the immune response in vivo, limits the development of immunological unresponsiveness and promotes maximal anti-tumor effects. Significantly prolonged survival was noted in tumor-carrying animals treated with Fab-SEA/Fab-IL-2 as compared with Fab-SEA or Fab-IL-2 alone. Combination therapy resulted in complete cure in 90% of tumor-bearing animals, whereas only 10% long-term survival was seen in Fab-SEA or Fab-IL-2-treated animals. Single Fab-SEA therapy induced a hyporesponsive state after 2 cycles of treatment. In contrast, the immune response after combination therapy was characterized by substantially augmented IFN-gamma and TNF-alpha production and strong CTL activity. Our data demonstrate that combined Fab-SEA and Fab-IL-2 therapy prolongs the immune response in vivo and induced long-term survival of more than 90% of the animals carrying the highly aggressive B16 melanoma.

Animals↗
PubMed1999

Tumor-growth inhibition with bispecific antibody fragments in a syngeneic mouse melanoma model: the role of targeted T-cell co-stimulation via CD28.

The ability of bispecific antibodies with anti-tumor x anti-CD3 specificity to mediate the killing of tumor cells by activated T cells has been demonstrated in many in vitro experiments. Moreover, long-term survival of lymphoma-bearing mice has been observed after treatment with such reagents. The therapeutic effect of bispecific antibodies in solid-tumor models has been less impressive, in particular if fragmented antibodies were used to avoid systemic T-cell activation by bispecific constructs binding to Fc-receptor-positive cells. Here we report that bispecific anti-tumor x anti-CD3-fragments markedly inhibit intraperitoneal as well as pulmonary tumor growth in mice inoculated with B16 melanoma cells, resulting in the long-term survival of animals. Therapeutic success critically depends on the number of recruitable effector cells at the site of tumor growth. A second bispecific construct triggering the co-stimulatory CD28-molecule on the T-cell surface increased tumor-cell killing in vitro and in vivo, despite rather low avidity of this reagent to mouse T cells. Finally, long-term-surviving animals showed improved survival after i.v. rechallenge with tumor cells, indicating that bispecific antibodies are capable of inducing long-lasting protective immunity.

Animals↗
PubMed1999

The distinct role of CD4+ and CD8+ T-cells during the anti-tumour effects of targeted superantigens.

To target T-cells to the tumour area we created a recombinant protein of the bacterial superantigen (SAg) Staphylococcal enterotoxin A (SEA) and the Fab-fragment of a tumour-reactive antibody. This antibody-targeted SAg immunotherapy therapy has been shown to be highly efficient, eliminating > 95% of the pulmonary metastasis in mice carrying established melanoma micrometastases. Earlier studies demonstrated that elimination of the C215-expressing B16-melanoma lung metastasis was dependent on interferon (IFN)-gamma release and expression of perforin. In the present study, therapeutic effector functions were analysed both locally at the tumour site and systemically in the spleen. In order to elucidate the role of each T-cell subset during Fab-SEA therapy, CD4 knock-out (KO) and CD8 KO mice were used. Tumour size reduction was statistically significant in Fab-SEA-based tumour therapy in both types of T-cell-deficient mice compared to wild-type mice. CD4 KO mice displayed a drastic reduction in the number of tumour-infiltrating macrophages and CD8+ T-cells. Therapy-induced accumulation of perforin-containing cells at the tumour site was significantly impaired in CD8 KO mice, and marginally in CD4 KO mice. Moreover, CD4 KO mice failed to produce substantial amounts of the tumour suppressive cytokine IFN-gamma. This is in sharp contrast to normal mice where a massive local release was recorded. CD8 KO mice displayed a spontaneous production of interleukin (IL)-4 and IL-10 locally in the tumour. Neither normal nor CD4 KO mice produced detectable levels of these Th-2-associated cytokines. The high level of IL-10 was demonstrated to inhibit Fab-SEA tumour therapy, since the therapeutic efficacy was significantly higher in IL-10 KO mice. These results illustrate the importance of a finely tuned cellular collaboration to regulate the various phases of an efficient anti-tumour immune response.

Animals↗
PubMed1999

Lymphoid hyperplasia in transgenic mice over-expressing a secreted form of the human interleukin-1beta gene product.

To evaluate the biological effects of over-expression of interleukin-1beta (IL-1beta) on the immune system we have generated transgenic mice, expressing the IL-1beta gene fused to a heterologous signal sequence under the control of the mouse immunoglobulin enhancer (Emu). A prominent hyperplasia and a disturbed microarchitecture of lymphoid tissues were observed in the transgenic mice. The CD4+ T cells in the hyperplastic lymphoid organs seemed to invade the majority of the lymphoid organs including B-cell restricted areas. Analysis of lymph node cells revealed an increased frequency of CD4+ CD44high CD62L- T cells and local secretion of IL-2 and IL-4, compatible with an elevated number of activated T cells. Furthermore, significant levels of human IL-1beta in sera and high concentrations of serum immunoglobulin G (IgG) were observed in the transgenic mice. The data suggest a role for IL-1beta in controlling lymphoid microarchitecture and, when over-expressed, breaking the threshold in T-helper-B-cell interaction.

Animals↗
PubMed1999

Rapid tyrosine phosphorylation of Lck following ligation of the tumor-associated cell surface molecule A6H.

We have recently described the A6H antigen as a novel 120-140 kDa molecule which is co-expressed on human peripheral blood T cells and renal cell carcinoma cells. Engagement of the A6H antigen results in co-stimulation of CD4+ T cells but it remained unknown how cross-talk between the A6H antigen and the TCR-CD3 complex takes place and which signaling pathway might be involved. Here we show that ligation of the A6H antigen with mAb induces tyrosine phosphorylation of the Lck protein tyrosine kinase (PTK). Co-ligation of the A6H antigen with CD3 resulted in augmented Lck phosphorylation and mitogenesis. In addition, A6H ligation induced an up-regulation of CD3-mediated phosphorylation of the 23 kDa high mol. wt form of TCR zeta and the zeta-associated protein, ZAP-70. Co-precipitation of Lck and ZAP-70 was only seen in T cells activated by combined A6H and anti-CD3 stimulation. In contrast, another Src family PTK, Fyn, was not affected by A6H ligation. In conclusion, we now demonstrate, for the first time, that A6H ligation triggers Lck phosphorylation, and that cross-talk between A6H and the TCR-CD3 complex involves Lck, ZAP-70 and the slow migrating isoform of TCR zeta. These results further suggests that A6H ligation is sufficient for triggering some of the early events in T cell activation, whereas full activation of the T cell, characterized by proliferation and cytokine production, requires co-ligation of the TCR-CD3 complex.

Antigens, Neoplasm↗
PubMed1999

Superantigens are presented by and activate thymocytes from infants.

A high percentage of human fetal and postnatal thymocytes express MHC class II molecules. This raises the possibility that human thymocytes in early life are able to present peptides to other immature T cells and thereby initiate thymic selection of these cells. Here we address this question by exposing newly harvested infant thymocytes to superantigen (Sag) which binds to the T-cell receptor and to MHC class II chains outside the peptide binding groove. The results show that the thymocytes are able to present Sag and to be activated to proliferation as well as apoptosis by Sag presented by other thymocytes. The absence of responses to Sag with mutations in class II binding sites showed that class II molecules were necessary for the responses, and very low expression of class II molecules on CD4-8- cells indicates that the demonstrated T-cell/T-cell interactions are confined to T-cell receptor-positive CD4+8+, CD4+8-, and CD4-8+ cells. These latter subsets were shown to be able to present Sag to each other. These findings suggest that class II+ thymocytes may participate in the selection of self-restricted T cells during a critical period in the shaping of the human immune system.

Antigen Presentation↗
PubMed1999

Treatment with tumor-reactive Fab-IL-2 and Fab-staphylococcal enterotoxin A fusion proteins leads to sustained T cell activation, and long-term survival of mice with established tumors.

C215Fab-IL-2 fusion protein, with full IL-2 and antigen binding activity, was produced in E. coli at high level (>50 mg/l). When co-administered with Fab-superantigen fusion protein (C215Fab-SEA) in mice strong and sustained T cell activation was observed. Combination treatment of mice carrying B16 melanoma transfected with C215 antigen was also more efficient than using C215Fab-SEA (p<0.01) or C215Fab-IL-2 alone (p<0.001). In a long-term survival experiment 5/12 mice having received combination treatment 5 days after i.v. inoculation of B16 cells survived >85 days. Improved therapeutic efficacy correlated with increased tumor infiltration by activated CD25+ T cells, indicating a T cell mediated mechanism.

Amino Acid Sequence↗
PubMed1998

In vivo anergized CD4+ T cells have defective expression and function of the activating protein-1 transcription factor.

The transcription factor activating protein-1 (AP-1) contributes significantly to the regulation of IL-2 gene expression during T cell activation and has been suggested to play a unique role in T cell anergy in vitro. In this study we have used the superantigen staphylococcal enterotoxin A to investigate the regulation of AP-1 in T cell anergy in vivo. Repeated injections of staphylococcal enterotoxin A induce a state of anergy in CD4+ T cells, characterized by reduced expression of IL-2 at mRNA and protein levels. The perturbed IL-2 response in anergic T cells correlated with reduced DNA binding activity of the transcription factors AP-1 and Fos/Jun-containing NF-AT. Using AP-1-luciferase reporter transgenic mice, we now demonstrate the lack of AP-1-dependent transcription. AP-1 activity is controlled by synthesis of its subunits Fos and Jun and by posttranslational phosphorylations. Analysis of Fos and Jun protein levels revealed no major differences in the expression of Jun proteins, but a marked decrease in c-Fos in anergic T cells. Experiments in transgenic mice overexpressing c-Fos (H2-c-fos) showed reconstituted AP-1 DNA binding. In contrast, the AP-1-driven transcription and IL-2 production remained suppressed. The Jun N-terminal kinase is known to play a critical role in regulating AP-1 trans-activation. Analyses of Jun N-terminal kinase demonstrated normal protein amounts, but reduced enzymatic activity, in anergic compared with activated CD4+ T cells. This suggests that in vivo anergized T cells have defects in the AP-1 pathway due to both reduced protein expression and perturbed posttranslational modifications.

Animals↗