Biomedical subjects

M Dohlsten

Publications and source records attributed to M Dohlsten.

At least 91 records · Page 5Linked to original sources

PubMed1995

Antibody-targeted superantigens induce lysis of major histocompatibility complex class II-negative T-cell leukemia lines.

CTLs bearing certain T-cell receptor V beta-regions are directed by the bacterial superantigen Staphylococcus enterotoxin A (SEA) to lyse MHC class II-positive cells. In order to extend superantigen-dependent cytotoxicity to MHC class II-negative carcinoma cells, covalent conjugates of superantigen and mAbs against surface markers of these cells have been used. We now describe a novel strategy which allows rapid selection of mAb suitable for superantigen targeting against MHC class II-negative tumor cells. A recombinant fusion protein of protein A and SEA binding to the mAbs CD7 or CD38 was able to mediate T cell-dependent lysis of MHC class II-negative Molt-4 and CCRF-CEM acute lymphatic leukemia cell lines. Lysis was dose dependent and correlated with E:T cell ratio. In contrast, SEA alone did not induce any significant lysis. In order to decrease the MHC class II affinity of the protein A-SEA complex, a point mutation was introduced into SEA (protein A-SEA mu9). The mutated fusion protein had similar potency as protein A-SEA against Molt-4 cells but was 100-fold less active against MHC class II-positive cells. Considering the efficiency and specificity of the mutated SEA protein interacting with mAb in targeting T lymphocytes against MHC class II-negative leukemia cells while only marginally affecting normal MHC class II-positive cells, we suggest the development of SEA-mAb fusion proteins as a potential adjuvant therapy of leukemias.

ADP-ribosyl Cyclase↗

PubMed1995

Distinct splicing of CD45 mRNA in activated rat gamma delta cytotoxic T lymphocytes.

Previous studies have demonstrated tumor- and allo-specific cytotoxic gamma delta T lymphocytes in rats. In this report we define the surface phenotype of these T cell receptor (TCR) gamma delta+ T cells and demonstrate distinct CD45 mRNA splicing in activated gamma delta cytotoxic T lymphocytes (CTL). gamma delta T lymphocytes in the blood and the peritoneal cavity were TCR alpha beta-CD3+CD8 alpha+CD45RC+ but expressed variable levels of LFA-1 molecules. Normal peritoneal gamma delta T lymphocytes, peritoneal gamma delta T cells from rats injected with the bacterial superantigen staphylococcal enterotoxin A (SEA) as well as gamma delta T lymphocytes in peripheral blood were all LFA-1low. Peritoneal gamma delta T cells from tumor-, and allo-sensitized rats were either LFA-1low or LFA-1high and specific cytotoxicity was highly enriched in the LFA-1high subset. No cytolytic activity against SEA-presenting cells was recorded in gamma delta T cells from SEA-injected rats. Different isoforms of CD45 in T cells are generated by alternative mRNA splicing of exons 4, 5, 6 (or A, B and C, respectively) and the recently described alternate exon 7. CD45 splicing in sorted gamma delta T cells was evaluated utilizing reverse transcription polymerase chain reaction. Normal peritoneal gamma delta T cells expressed exon(578), exon(678), exon(78) and the extensively spliced exon(8) variant. Peritoneal gamma delta T cells from rats sensitized with irradiated syngeneic tumor cells, allogeneic cells or bacterial superantigen SEA as well as gamma delta T lymphocytes in peripheral blood contained the full-length exon(45678), as well as the exon(5678), exon(578), exon(678) and exon(78) splicing products. Notably, the exon(8) variant was also seen in peritoneal gamma delta T cells of SEA-sensitized rats. Sorted tumor-specific LFA-1high gamma delta CTL expressed exon(45678), exon(5678), exon(578), exon(678) and exon(78) CD45 splicing products whereas the non-cytolytic LFA-1low gamma delta T cell subset also contained exon(8) variant. In summary, it is concluded that antigen-specific TCR gamma delta+ CTL express high levels of LFA-1 and that the splicing machinery in these cytolytic cells favors expression of the exon(45678) and exon(5678) CD45 splicing products whereas the exon(8) variant is lost. TCR alpha beta+ CTL express high levels of LFA-1 but are devoid of the full-length exon(45678) splicing product. The different CD45 splicing patterns found in alpha beta CTL and gamma delta CTL indicate different molecular requirements in respect to CD45 during activation and differentiation of these T lymphocyte subsets.

Alternative Splicing↗

PubMed1995

Immunotherapy of human colon cancer by antibody-targeted superantigens.

T lymphocytes generally fail to recognize human colon carcinomas, suggesting that the tumour is beyond reach of immunotherapy. Bacterial superantigens are the most potent known activators of human T lymphocytes and induce T cell cytotoxicity and cytokine production. In order to develop a T-cell-based therapy for colon cancer, the superantigen staphylococcal enterotoxin A (SEA) was given tumour reactivity by genetic fusion with a Fab fragment of the monoclonal antibody C242 reacting with human colon carcinomas. The C242Fab-SEA fusion protein targeted SEA-reactive T cells against MHC-class-II-negative human colon carcinoma cells in vitro at nanomolar concentrations. Treatment of disseminated human colon carcinomas growing in humanized SCID mice resulted in marked inhibition of tumour growth and the apparent cure of the animals. Therapeutic efficiency was dependent on the tumour specificity of the fusion protein and human T cells. Immunohistochemistry demonstrated massive infiltration of human T cells in C242Fab-SEA-treated tumours. The results merit further evaluation of C242Fab-SEA fusion proteins as immunotherapy in patients suffering from colon carcinoma.

Animals↗

PubMed1995

Enhanced and prolonged efficacy of superantigen-induced cytotoxic T lymphocyte activity by interleukin-2 in vivo.

The bacterial superantigen, staphylococcal enterotoxin A (SEA) activates T cells with high frequency and directs them to lyse MHC-class-II-expressing cells in superantigen-dependent cell-mediated cytotoxicity (SDCC). Treatment of mice with SEA induced strong CD8+ T-cell(CTL)-mediated SDCC, as well as abundant cytokine production from CD4+ and CD8+ T cells. However, both cytotoxicity and cytokine release were transient. In contrast, combined treatment with SEA and recombinant interleukin-2 (rIL-2) increased peak levels and maintained CTL activity. These effects were concomitant with an increased number of SEA-reactive V beta 11+ T cells. Both the CD4+ and CD8+ populations contained higher frequencies of cells expressing IL-2 receptor (IL-2R) alpha beta, which suggests that continuous IL-2R signaling preserves its high expression and subsequently prevents loss of growth factor signals necessary for expansion of T cells. Although IL-2R expression was increased among both CD4+ and CD8+ cells, only the cytotoxic function of CTL, but not cytokine production from either CD4 or CD8, was augmented. These findings demonstrate that treatment with rIL-2 potentiates superantigen-induced cytotoxicity and maintains high CTL activity. rIL-2 might therefore be useful in improving superantigen-based tumor therapy.

Animals↗

PubMed1995

Superantigen-staphylococcal-enterotoxin-A-dependent and antibody-targeted lysis of GD2-positive neuroblastoma cells.

Superantigens such as the staphylococcal enterotoxin A (SEA) are among the most potent T cell activators known. They bind to major histocompatibility complex (MHC) class II molecules and interact with T cells depending on their T cell receptor (TCR) V beta expression. Superantigens also induce a variety of cytokines and trigger a direct cytotoxic effect against MHC-class-II-positive target cells. In order to extend superantigen-dependent cell-mediated cytotoxicity (SDCC) to MHC-class-II-negative neuroblastoma cells, SEA was linked to the anti-ganglioside GD2 human/mouse chimeric monoclonal antibody (mAb) ch14.18. Ganglioside GD2 is expressed on most tumours of neuroectodermal origin but is expressed to a lesser extent on normal tissues. The linkage of ch14.18 to SEA was achieved either with a protein-A-SEA fusion protein or by chemical coupling. Both constructs induced T-cell-mediated cytotoxicity towards GD2-positive neuroblastoma cells in an effector-to-target(E:T)-ratio- and dose-dependent manner in vitro. To reduce the MHC class II affinity of SEA, a point mutation was introduced in the SEA gene (SEAm9) that resulted in 1000-fold less T cell killing of MHC-class-II-expressing cells as compared to native SEA. However, a protein-A-SEAm9 fusion protein mediated cytotoxicity similar to that of protein-A-SEA on ch14.18-coated, MHC-class-II-negative neuroblastoma cells. Taken together, these findings suggest that superantigen-dependent and monoclonal-antibody-targeted lysis may be a potent novel approach for neuroblastoma therapy.

Antibodies, Monoclonal↗

PubMed1995

A novel co-stimulatory T cell antigen co-expressed on renal cell carcinoma.

The A6H mAb raised primarily against human renal cell carcinoma (RCC) has previously been shown to bind strongly to RCC, to some degree to colon carcinoma but only marginally to a variety of normal tissues. Immunohistochemical analysis or RCC tissues containing tumor-infiltrating lymphocytes revealed that A6H stained both tumor cells and lymphocytes. FACS analysis of human peripheral blood cells demonstrated that A6H stained both tumor cells and lymphocytes. FACS analysis of human peripheral blood cells demonstrated that A6H mAb stained 85-90% of both CD4+ and CD8+ T cells, but not granulocytes, monocytes, NK cells or B cells. Furthermore, 85-90% of naive and memory T helper cells were stained with A6H suggesting that the A6H mAb defines unique subsets within these T cell populations. Dual staining showed that A6H mAb bind to an antigen that is clearly distinct from other cell surface molecules on T cells, including CD28, CD29, CD26, CD44 and ICAM-2. A6H mAb binding induced a second signal in anti-CD3 mAb activated T cells, resulting in cell proliferation, IL-2 receptor expression and vigorous production of IFN-gamma and TNF, and production of minor amounts of IL-2. Immunoprecipitation with A6H mAb indicated a molecular weight of 120-140 kDa on both T cells and RCC. We suggest that the A6H mAb defines a unique T cell surface antigen which is involved in signal transduction and is expressed on subsets of human T cells. The co-expression of A6H on T cells and tumor cells suggests a possible function related to common properties of these cells.

Antibodies, Monoclonal↗

PubMed1995

Expression of CD11a and CD45R isoforms defines distinct subsets of CD8+ TCR alpha beta and TCR gamma delta CTL in vivo.

Intraperitoneal immunization of rats with a syngeneic lymphoma and allogeneic leukocytes induced enrichment of antigen-selective TCR alpha beta+ and TCR gamma delta+ CTL. The peritoneal cavity seems to be a suitable site for enrichment of antigen-selective CTL, since the induced effector cells executed strong cytotoxicity without any requirement for in vitro reactivation. Tumor-selective CTL expressed high cell surface levels of CD45RC, allogeneic CTL expressed a variable level of CD45RC, while SAg-reactive CTL had low CD45RC expression. CTL with tumor and allogeneic selectivity as well as SAg-induced CTL all expressed the LFA-1high phenotype, suggesting that upregulation of LFA-1 is a hallmark for in vivo activated CTL. RT-PCR analyses showed that all CD8+TCR alpha beta+ CTL lost expression of CD45R exon 4 mRNA, which is compatible with the view that effector/memory T cells are CD45RA-. In contrast, TCR gamma delta+ CTL retained the CD45RA phenotype but showed a down-regulation of CD45R exon 8 mRNA. Since isoforms of the CD45 tyrosine phosphatase have been reported to differentially affect T-cell activation, the unique CD45R splice pattern observed in TCR alpha beta+ and TCR gamma delta+ CTL implies that CD45R may influence the TCR signal transduction distinctly in various effector CTL subsets.

Animals↗

PubMed1995

T cell activation pathways: B7, LFA-3, and ICAM-1 shape unique T cell profiles.

Two signals are required for induction of cell proliferation and cytokine production in resting T cells. Occupancy of the T cell receptor by antigen/MHC complexes delivers the first signal to the T cell, while the second signal is provided by interaction with costimulatory ligands on APC. CD2, LFA-1, and CD28 are the major costimulatory and adhesive molecules on T cells and bind to the LFA-3, ICAM-1 and B7 ligands, respectively, on APC. LFA-3 plays a central role for naive and memory T helper cells during the early phase of an immune response. The LFA-3/CD2 pathway initiates strong antigen-independent cell adhesion, substantial expansion of naive T helper cells, and induction of large amounts of IFN-gamma in memory cells. The release of IFN-gamma may upregulate expression of ICAM-1 and B7 on APC and allows multiple adhesion pathways to amplify the immune response. The LFA-1/ICAM-1 pathway stimulates adhesion and cell proliferation more efficiently in memory T helper cells than in naive cells. Further, the results suggest that naive T helper cells express functionally inactive LFA-1 molecules on the cell surface, which may have a physiological role in keeping these cells in a resting state. B7 costimulation superinduces IL-2 production in both naive and memory T helper cells and generates long-lasting cell proliferation. This permits transition from an autocrine to a paracrine immune response. Coexpression of B7/LFA-3 provides an optimal APC function and enables a vigorous T cell response to minute amounts of antigen. AP-1 and NF-kappa B transcription factors are involved in the induction of several cytokine gene promoters and play a central role in the regulation of IL-2 gene transcription. LFA-3 costimulation only moderately enhances AP-1 DNA-binding activity and does not influence the NF-kappa B activity induced by TCR engagement, whereas B7 costimulation induces large amounts of NF-kappa B and AP-1 activity in T helper cells. The costimulatory ligands represent a family of adhesion molecules with considerable redundancy. Interfamily redundancy of LFA-3, B7, and ICAM ligands offers an opportunity to regulate distinct T cell response profiles in various microenvironments at separate time points of an immune response.

Animals↗

PubMed1995

Antibodies are capable of directing superantigen-mediated T cell killing of chronic B lymphocytic leukemia cells.

The bacterial superantigen staphylococcal enterotoxin A (SEA) is a highly potent activator of cytotoxic T cells when presented on MHC class II molecules of target cells. Our earlier studies showed that such SEA-directed T cells efficiently killed chronic B lymphocytic leukemia (B-CLL) cells. With the ultimate goal to replace the natural specificity of SEA for MHC class II molecules with the specificity of a monoclonal antibody (mAb), we initially made a mutated protein A-SEA (PA-SEAm) fusion protein with > 100-fold reduced binding affinity for MHC class II compared to native SEA. The fusion protein was successfully used to direct T cells to B-CLL cells coated with different B lineage specific (CD19, CD20) or associated (CD37, CD40) mAbs. The PA-SEAm protein was 10-100-fold more potent against mAb coated compared to uncoated HLA class II+ B-CLL cells. No correlation was seen between the amount of mAb bound to the cell surface and sensitivity to lysis. Preactivation of B-CLL cells by phorbol ester increased their sensitivity, and lysis was dependent on ICAM-1 molecules. However, no preactivation of the target cells was needed when a cocktail of two or four mAbs was used. Circulating leukemia and spleen cells were equally well killed. We conclude that the natural target specificity of SEA, MHC class II, can be reduced by mutagenesis and novel binding specificity can be introduced by linkage to tumor reactive mAbs. Our findings encourage the construction of recombinant SEA mutant fusion proteins for specific T cell therapy of hematopoietic tumors such as B-CLL.

Antibodies, Monoclonal↗

PubMed1994

Costimulation of human CD4+ T lymphocytes with B7 and lymphocyte function-associated antigen-3 results in distinct cell activation profiles.

This study describes the distinct roles of B7 and LFA-3 in the regulation of T cell responses. Activation of CD4+ T cells with Chinese hamster ovary (CHO)-DR4/B7 and CHO-DR4/LFA-3 cells that present the superantigen staphylococcal enterotoxin A resulted in significant T cell proliferation and substantial production of TNF and IFN-gamma. Strong IL-2 production was recorded in B7-costimulated, but not LFA-3-costimulated, cultures. The presence of B7 induced a more vigorous and prolonged proliferative T cell response compared with LFA-3 costimulation. In contrast, LFA-3 was more efficient than B7 in mediating cell adhesion of CD4+ T cells. Costimulation with the CHO-DR4/B7/LFA-3 triple transfectant resulted in enhanced cell adhesion, proliferation, and cytokine production compared with either DR4/B7 or DR4/LFA-3 alone. Optimal production of IL-2 by naive and memory CD4+ T cells was seen only when cells were costimulated with B7, whereas IFN-gamma production was induced in memory cells by both LFA-3 and B7. The Jurkat T cell line responded to CHO-DR4/B7/LFA-3 in a manner similar to peripheral blood CD4+ T cells. Reverse transcriptase-PCR analysis of Jurkat cells stimulated with staphylococcal enterotoxin E and the different CHO transfectants revealed that the cooperative effect of B7 and LFA-3 on IL-2 production was also seen at the mRNA level. The large amounts of IL-2 produced by B7 costimulation indicate a paracrine function of the B7/CD28 pathway, whereas the LFA-3/CD2 pathway provides strong adhesion and may facilitate autocrine T cell expansion. Combined expression of the B7 and LFA-3 molecules seems to provide an optimal Ag-presenting function that ensures strong adhesion and optimal signal transduction.

Animals↗

PubMed1994

Monoclonal antibody-superantigen fusion proteins: tumor-specific agents for T-cell-based tumor therapy.

The bacterial superantigen staphylococcal enterotoxin A (SEA) is an extremely potent activator of T lymphocytes when presented on major histocompatibility complex (MHC) class II molecules. To develop a tumor-specific superantigen for cancer therapy, we have made a recombinant fusion protein of SEA and the Fab region of the C215 monoclonal antibody specific for human colon carcinoma cells. SEA as part of a fusion protein showed a > 10-fold reduction in MHC class II binding compared to native SEA, and accordingly, the affinity of the FabC215-SEA fusion protein for the C215 tumor antigen was approximately 100-fold stronger than to MHC class II molecules. The FabC215-SEA fusion protein efficiently targeted T cells to lyse C215+ MHC class II- human colon carcinoma cells, which demonstrates functional substitution of the MHC class II-dependent presentation of SEA with tumor specificity. Treatment of mice carrying B16 melanoma cells expressing a transfected C215 antigen resulted in 85-99% inhibition of tumor growth and allowed long-term survival of animals. The therapeutic effect was dependent on antigen-specific targeting of the FabC215-SEA fusion protein, since native SEA and an antigen-irrelevant FabC242-SEA fusion protein did not influence tumor growth. The results suggest that Fab-SEA fusion proteins convey superantigenicity on tumor cells, which evokes T cells to suppress tumor growth.

Antibodies, Monoclonal↗

PubMed1994

Combined activation of murine lymphocytes with staphylococcal enterotoxin and interleukin-2 results in additive cytotoxic activity.

This report demonstrates that in vitro activation of murine spleen cells with interleukin-2 (IL-2) or the bacterial superantigen staphylococcal enterotoxin A (SEA) results in different patterns of activation and function of cytotoxic cells. Lymphokine-activated killer activity and antibody-dependent cellular cytotoxicity (ADCC) are mainly mediated by IL-2 activated natural killer (NK) cells. SEA is the most powerful T cell mitogen known so far and retargets cytotoxic T lymphocytes (CTL) to tumors expressing major histocompatibility complex (MHC) class II in staphylococcal-enterotoxin-dependent cellular cytotoxicity (SDCC). Culture of mouse spleen cells with SEA led to expansion and activation of T cells, which demonstrated strong SDCC activity and some NK-like cytotoxicity after 5 days in culture. Cell sorting revealed that both CD8+ and CD4+ T cells mediated SDCC but the former were more effective. Phenotypic analysis showed that SEA preferentially stimulated and expanded T cells expressing T cell receptor V beta 11, in particular CD8+ T cells. Combined activation with SEA and IL-2 resulted in simultaneous induction of T and NK cell cytotoxicity. Moreover, IL-2 had additive effects on SEA-induced SDCC. Combined treatment with SEA and IL-2 might therefore be an approach to induce maximal cytotoxicity against tumors and to recruit both T and NK cells in tumor therapy.

Animals↗

PubMed1994

Different types of in vivo induced cytolytic T lymphocytes are all LFA-Ihigh and lack CD45 exon 4 (CD45RA) but show distinct CD45RC profiles.

Due to alternate mRNA splicing of exons 4, 5 and 6 (or A, B and C respectively), the CD45 cell surface glycoprotein is structurally heterogeneic in lymphoid cells of different lineage or stage of activation. Previous studies show that in vivo induced allo- and superantigen reactive rat cytolytic T lymphocytes (CTL) preferably belong to the CD45RClow subset, whereas tumour selective CTL express high amounts of CD45RC cell surface molecules. In this paper, reverse transcription polymerase chain reaction technique (RT-PCR) was utilized to evaluate CD45 isoform expression of rat lymphoid cells and in vivo activated rat CTL with distinct specificity and CD45RC profile. Cells from lymphoid organs expressed six CD45 mRNA isoforms, exon(45678), exon(5678), exon (578), exon(678), exon(78) and a novel extensively spliced exon(8) variant. In vivo activated TCR alpha beta + CD8+ cells sorted as CD45RClow expressed exon(78), exon(578) and exon(8), whereas TCR alpha beta + CD8+CD45RChigh cells expressed exon(78), exon(578), exon(5678) and full-length exon(45678). Triple-colour staining indicated high expression of LFA-1 in the cytotoxic CD45RCintermediate and CD45RClow cells, and low expression of LFA-1 in CD45RChigh non-cytotoxic cells from allo- and superantigen activated rats. In contrast, tumour activated TCR alpha beta +CD45RChigh cells were divided in LFA-1high and LFA-1low subsets, and sorting of these subsets revealed that tumour-selective cytotoxicity was confined to the LFA-1high effector cell subset. Furthermore, it was evident that the LFA-1high effector cell subset expressed high levels of exon(5678), exon(578) and exon(78) isoforms and, in contrast to the LFA-1low subpopulation, lacked expression of exon 4 containing full-length CD45 mRNA transcript.(ABSTRACT TRUNCATED AT 250 WORDS)

Alternative Splicing↗

PubMed1994

Superantigens anergize cytokine production but not cytotoxicity in vivo.

We have investigated the effect of the superantigen staphylococcal enterotoxin A (SEA) on the balance between T-cell response and non-responsiveness in T-cell receptor (TcR) V beta 3 transgenic mice. One injection of SEA resulted in a substantial activation of TcR V beta 3+ cells, whereas T cells from mice injected with repeated doses of SEA displayed a diminished response to a subsequent in vitro challenge. The reduced responsiveness became apparent when SEA was injected multiple times with short intervals. Proliferation and cytokine production in anergized T cells were severely reduced when stimulated with SEA in vitro, whereas cytotoxic T lymphocyte (CTL) activity remained unaffected. The dichotomy between these functions was examined in vitro with respect to different T-cell subsets. The total number of CD4+ T cells was reduced in the hyporesponsive spleens, compatible with cell deletion. The remaining CD4+ TcR V beta 3+ T cells showed anergy of all tested functions and did not respond to exogenous interleukin-2 (IL-2). In contrast, there was an expansion of CD8+ TcR V beta 3+ T cells with an intact cytotoxic activity. The in vitro proliferation and production of cytokines in the CD8+ compartment was impaired, but could be partially restored in the presence of exogenously added IL-2. Analysis of the cytokine response to SEA in vivo showed that IL-2 and tumour necrosis factor (TNF) were mainly produced by CD4+ T cells, while interferon-gamma (IFN-gamma) was predominantly released by CD8+ T cells. Induction of anergy resulted in a reduction of IL-2 and TNF mRNA levels, frequencies of producing cells as well as serum protein content. In contrast, there was only a moderate influence on the IFN-gamma level in vivo. The results suggest that SEA-induced hyporesponsiveness involves CD4+ cell deletion and a failure to produce cytokines in the remaining CD4+ T-cell compartment, while IFN-gamma production and cytotoxicity in the CD8+ T-cell compartment stay relatively intact.

Animals↗

PubMed1994

Alloselective natural killer cells are activated by allogeneic heart transplantation and can potentially play a functional role as classic CTLs.

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Animals↗

PubMed1993

Isolation, partial characterization, and molecular cloning of a human colon adenocarcinoma cell-surface glycoprotein recognized by the C215 mouse monoclonal antibody.

The monoclonal antibody C215 (IgG2a) was obtained by the immunization of BALB/c mice with the human colon adenocarcinoma cell line COLO 205 and used in the targeting of colorectal carcinomas. The partial characterization and purification of the C215 target molecule from solubilized COLO 205 membranes indicated that it is an integral membrane glycoprotein of the non-mucin type. The denatured antigen appeared as a major 40-kDa form in Western blots after SDS-polyacrylamide gel electrophoresis and migrated as a monomeric 36-kDa species after the reductive cleavage of intramolecular disulfide bridges. Using a five-step procedure, the antigen was purified 4,300-fold from COLO 205 tumors raised in nude mice to a homogeneity of 95% when assessed by capillary electrophoresis. Removal of N-linked carbohydrate by peptide:N-glycosidase treatment did not affect the visualization of the purified antigen in immunoblots but resulted in a faster migration in the SDS gels. The amino acid sequence was partially determined. Seventeen contiguous NH2-terminal amino acids were identified and coincided exactly with residues 82-98 of the GA733-2 protein cloned by Szala et al. (Szala, S., Froehlich, M., Scollon, M., Kasai, Y., Steplewske, Z., Koprowski, H., and Linnenback, A. J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3542-3546). Therefore, the predicted amino acid sequence of this protein was used to prepare overlapping synthetic peptides that cover the entire extracellular domain in order to identify the C215 epitope. A likely epitope, close to the NH2 terminus and corresponding to the first distinct hydrophilic stretch after the putative signal sequence, was identified in a peptide enzyme-linked immunosorbent assay. Moreover, GA733-2 cDNA was used for the cloning of the C215 protein from COLO 205 cells and the subsequent transfection to K36.16 mouse T cell leukemia cells. The transfected cells were C215 reactive in fluorescence-activated cell sorter analysis, and a 42 kDa band was visualized in Western blots under both non-reducing and reducing conditions. Our findings indicate a close relationship between the C215 antigen and other members of the GA-733 family, some of which are currently being used as targets in clinical trials with monoclonal antibodies. The mammalian expression system described here will enable further studies into the biological role of this protein and the construction of animal models in order to develop optimal therapeutic strategies.

Adenocarcinoma↗

PubMed1993

Efficient killing of chronic B-lymphocytic leukemia cells by superantigen-directed T cells.

In vitro studies have indicated that chronic lymphocytic leukemia of B-cell origin (B-CLL) is resistant to cytotoxic effector lymphocytes such as natural killer and lymphokine activated killer (LAK) cells. We show here that B-cell cells are sensitive to Staphylococcal enterotoxin (SE) A-directed T-cell killing. Activation of the target cells by phorbol ester (tetradecanoyl phorbol acetate, [TPA]) greatly enhances their sensitivity to lysis. In SE-dependent cellular cytotoxicity (SDCC), members of the SE superantigen family form a bridge between T cells and target cells expressing major histocompatability complex class II molecules. Binding of SEA to the T-cell-receptor V beta region induces a strong cytotoxic capacity and cytokine production. Cells from 9 B-CLL patients were cultured in the presence or absence of TPA and used as targets in a 4-hour SDCC assay using an allogeneic T-cell line as effector. At an effector:target cell ratio 30:1, 70% to 80% of TPA-induced B-CLL cells were killed. Even at the effector:target ratio of 3:1, 47% +/- 6% of TPA-activated B-cell cells were lysed compared with 13% +/- 2% of resting cells (P < .001). A T-cell line established from a B-CLL patient killed autologous tumor cells as efficiently as allogeneic effectors. SEA-directed T cells were far more lytic to B-CLL cells compared with LAK cells or lectin (phytohemagglutinin-directed T cells. Mechanisms of SDCC lysis were investigated. Effector plus target cell supernatants contained high levels of tumor necrosis factor (TNF)-alpha and interferon-gamma, but these supernatants were not directly toxic to B-CLL cells in short term culture. High concentrations of recombinant TNF-alpha or TNF-beta had no lytic effect. Addition of neutralizing anti-TNF-alpha and anti-TNF-beta antibodies into the SDCC assay did not inhibit SEA-directed T-cell killing. TPA-activated B-CLL cells showed a 1.2- to 13-fold increased expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-associated antigen (LFA)-1, and LFA-3, whereas expression of HLA class II molecules increased up to 5 times. The expression of CD72, CD40, and BB-1/B7 increased 1.8 to 4.5 times. The role of these surface molecules in SDCC was analyzed in blocking experiments with monoclonal antibodies. Antibodies to ICAM-1, CD18, and HLA-DR abolished the cytotoxicity, and a substantial reduction was seen with antibody to CD72.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗

PubMed1993

Superantigen-induced cytokines suppress growth of human colon-carcinoma cells.

We have recently demonstrated that the superantigen staphylococcal enterotoxin A (SEA) conjugated to colon-carcinoma-reactive monoclonal antibodies (MAbs) directs T cells to lyse human colon-carcinoma cells, representing a potential novel tumor therapy. To further analyze the mechanism of antitumor effects of superantigen-activated T cells, we compared the activity of free and MAb-conjugated SEA in a long term in vitro co-culture assay of human peripheral-blood mononuclear cells (PBMC) and colon-carcinoma cell lines. Activation of resting T lymphocytes with SEA conjugated to the colon-carcinoma-reactive MAb C215 or free SEA resulted in strong inhibition of the growth of all studied colon-carcinoma cell lines. The growth of WiDr colon-carcinoma cells was unaffected by the presence of unactivated mononuclear cells, whereas addition of pM concentrations of SEA or C215-SEA conjugate completely suppressed tumor-cell growth. The suppressive effect was mediated by both CD4+ and CD8+ T cells and required the presence of MHC-Class II+ monocytes. The inhibition of tumor-cell growth was to a large extent mediated by soluble factors present in supernatants from SEA- or C215-SEA-activated mononuclear cells. Quantitation of cytokine mRNA in SEA-activated mononuclear cells by the reverse transcriptase-polymerase chain reaction (RT-PCR) revealed strong induction of mRNA encoding the cytokines IL-1 alpha, IL-1 beta, IL-2, IL-6, TNF-alpha, TNF-beta and IFN-gamma. The use of cytokine-specific MAb demonstrated that IFN-gamma was of major importance for the tumor-growth-inhibitory activity in supernatants of SEA-activated lymphocytes. Addition of recombinant cytokines to WiDr colon-carcinoma cells showed that TNF-alpha was able to act synergistically with IFN-gamma to suppress tumor-cell growth. The local production of tumor-suppressive cytokines induced by MAb-targeted superantigens is likely to be of particular relevance for inhibition of the growth of tumor cells not expressing the targeted tumor-associated antigen.

Antibodies, Monoclonal↗