Mikael Dohlsten

CHARACTERIZATION OF TWO DISTINCT MHC CLASS II BINDING SITES IN THE SUPERANTIGEN STAPHYLOCOCCAL ENTEROTOXIN A

Bacterial superantigens (SAgs) are potent activators of T lymphocytes and play a pathophysiological role in Gram‐positive septic shock and food poisoning. To characterize potential MHC class II binding sites of the bacterial SAg staphylococcal enterotoxin (SE) A, we performed alanine substitution mutagenesis throughout the C‐terminus and at selected sites in the N‐terminal domain. Four amino acids in the C‐terminus were shown to be involved in MHC class II binding. Three of these amino acids, H225, D227 and H187, had a major influence on MHC class II binding and appeared to be involved in coordination of a Zn2+ ion. Alanine substitution of H225 and D227 resulted in a 1000‐fold reduction in MHC class II affinity. Mutation at F47, which is equivalent to the F44 previously shown to be central in the MHC class II binding site of the SAg, SEB, resulted in a 10‐fold reduction in MHC class II affinity. The combination of these mutations in the N‐ and C‐terminal sites resulted in a profound loss of activity. The perturbation of MHC class II binding in the various mutants was accompanied by a corresponding loss of ability to induce MHC class II‐dependent T cell proliferation and cytotoxicity. All of the SEA mutants were expressed as Fab‐SEA fusion proteins and found to retain an intact T cell receptor (TCR) epitope, as determined in a mAb targeted MHC class II‐independent T cell cytotoxicity assay.(ABSTRACT TRUNCATED AT 250 WORDS)

The crystal structure of staphylococcal enterotoxin type D reveals Zn2+-mediated homodimerization.

Bacterial superantigens, including the staphylococcal enterotoxins, are the most potent activators of T cells known and have been suggested as a causative factor in Gram‐positive shock in humans. Staphylococcal enterotoxin D (SED) is dependent upon Zn2+ for high affinity interactions with MHC class II molecules and thus SED was co‐crystallized with Zn2+. The crystal structure of SED has been determined in two different space groups, at 2.3 and 3.0 A resolution respectively. The three‐dimensional structure of SED is similar to structures of other bacterial superantigens, although this study has revealed that SED has the unique capability of forming dimers in the presence of Zn2+. The high affinity Zn2+ site used in dimer formation is located on the surface of the beta‐sheet in the C‐terminal domain. Two bound metal ions are coordinated by residues from both molecules in the dimer interface and thus contribute directly to formation of the dimer. A second Zn2+ site is located on the surface close to the domain interface of the molecule. The unique feature of SED in forming a Zn2+‐dependent homodimer seems to facilitate novel and biologically relevant multimeric interactions with MHC class II molecules, as shown by the induction of cytokine mRNA in human monocytes when exposed to SED and SED mutants.

Histamine acts directly on human T cells to inhibit interleukin-2 and interferon-γ production

Abstract Histamine acts directly on human T cells to inhibit lymphokine production without the involvement of accessory cells. Histamine inhibits the production of interleukin-2 (IL-2) and interferon-γ (IFN-γ) by purified human peripheral T cells activated in the presence of either intact monocytes or metabolically inactive fixed Raji and U698 cells as accessory cells. Purified T cells do not respond more than marginally to staphylococcal enterotoxin A (SEA) or phytohemagglutinin (PHA) in the absence of accessory cells. However, activation by the phorbol ester PMA in conjunction with either PHA or the calcium ionophore A23187 induces large amounts of IFN-γ and IL-2. Histamine suppresses the lymphokine production in these pure T-cell cultures to a similar extent as in monocyte-containing cultures. Histamine is also shown to suppress DNA synthesis by purified T cells cultivated at a low cell density, eliminating any possible involvement of small numbers of contaminating accessory cells. In vitro preactivated T cells are shown to retain their capacity to respond to histamine when stimulated by PMA and A23187 or by mitogen in the presence of Raji cells. The conclusion that histamine acts directly on T cells and does not require accessory cells to induce suppression is further confirmed by the demonstration that IL-2 production by the human T-cell leukemia line Jurkat was significantly suppressed by histamine in a H-2 receptor-restricted manner.

Histamine inhibits interferon-γ production via suppression of interleukin 2 synthesis

Histamine, a modulator of various immune functions, inhibits the production of interleukin 2 (IL-2) and interferon-gamma (IFN-gamma) by polyclonally activated human blood mononuclear cells. The histamine-induced inhibition of IFN-gamma synthesis can be completely eliminated by the addition of recombinant IL-2. The IFN-gamma synthesis by T8+ lymphocytes is highly dependent on IL-2 supplied either by the IL-2 producing T4+ lymphocytes or through exogenous addition of recombinant IL-2. It is concluded that histamine acts primarily on the interleukin 2 synthesis by the T4+ lymphocytes and as a consequence of this inhibition, interferon-gamma production is reduced.

CTLA-4 Ligation Suppresses CD28-induced NF-κB 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-κB 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-κB and AP-1 family members were similar, irrespective of stimulation conditions. Analyses of the NF-κB regulator IκB-α revealed similar levels of IκB-α protein in the preparations. However, a reduced phosphorylation of IκB-α 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.

Histamine Inhibits Interleukin 1 Production by Lipopolysaccharide-Stimulated Human Peripheral Blood Monocytes

Histamine inhibited the production of interleukin 1 (IL‐1) induced by lipopolysaccharide (LPS) in cultures of purified human peripheral blood monocytes. The effect of histamine on IL‐1 production was dose‐dependent and significant at histamine concentrations of 10−4‐10−5m. The histamine H2 receptor agonists dimaprit and 4‐methylhisiamine, hut not the H1 receptor agonists 2‐pyridylethylamine. aminoethylthiazole and 2‐methylhistamine, modulated the IL‐1 production in a similar manner to histamine. The inhibitory effects of histamine could be reversed by the H2 receptor antagonist cimetidine but not by the H1 receptor antagonist mepyramine. This indicates that the inhibitory effects of histamine on LPS‐induced IL‐1 production are mediated through H2 receptors on human peripheral blood monocytes.

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.

Staphylococcal enterotoxins direct and trigger CTL killing of autologous HLA-DR+ mononuclear leukocytes and freshly prepared leukemia cells

Attempts have been made to induce cytolytic T cells to kill target cells that do not express the appropriate target molecules by crosslinking the T cells and the target cells in various ways. One successful strategy has been to use heteroconjugates or bispecific monoclonal antibodies reacting with T cell molecules with activating properties (e.g., mab directed to CD3/TCR) and target cell surface antigens. In this report we show that Staphylococcal enterotoxins (SE) direct human T lymphocytes to execute cytotoxicity toward MHC class II-expressing Raji cells, but not against MHC class II-deficient Raji mutant RJ 2.2.5. Both HLA-DR+ and HLA-DR- effector T lymphocytes are effective in the killing of Raji cells coated with SE. The Staphylococcal enterotoxin-dependent cell-mediated cytotoxicity (SDCC) is a rapid T lymphocyte-mediated cytolytic mechanism killing the targets within an hour of incubation. HLA-DR+ target cells are sensitized to be killed within minutes of incubation with picomolar concentrations of SE. SE-sensitized Raji cells remain targets for SDCC after overnight culture at 37 degrees C, demonstrating that the sensitive state is relatively stable. SEA- and SEB-selective cytolytic T cell lines were established to illustrate the clonal variability of SDCC effectors with respect to SE specificity. We also demonstrate that autologous monocytes and activated T lymphocytes as well as B lymphocytes and freshly prepared HLA-DR+ leukemic cells are excellent targets in SDCC.

Superantigen-based tumor therapy: in vivo activation of cytotoxic T cells.

We have recently demonstrated that the superantigen staphylococcal enterotoxin A (SEA) targets in vitro activated cytotoxic T lymphocytes against tumor cells expressing major histocompatibility complex (MHC) class II antigens. In this report we analyze the use of SEA as an immunoactivator in vivo. Treatment of mice with SEA activated a fraction of CD3+ T cells apparently as a function of their T cell receptor Vβ expression. SEA induced interleukin-2 receptor expression and proliferation in both CD4+ and CD8+ T cells. This proliferative response was dose-dependent (0.1 – 100 µg/mouse), peaked during day 1 after treatment and declined to background levels within 4 days. The cytotoxic response, measured as cytotoxicity to SEA-coated MHC class II+ target cells (staphylococcal-enterotoxin-dependent cell-mediated cytotoxicity, SDCC), was maximal at a dosage of 1 µg SEA/mouse. The SDCC was confined to the CD8+ T cell compartment, peaked 2 days after treatment and declined to background levels within 4 days. A second injection of SEA on day 5 after the first SEA treatment resulted in SDCC function with kinetics and magnitude identical to that seen after one injection. These results pave the way for the use of SEA in the treatment of MHC class II+ tumors.

The Co-crystal structure of staphylococcal enterotoxin type A with Zn2+ at 2.7 A resolution. Implications for major histocompatibility complex class II binding.

Superantigens form complexes with major histocompatibility complex (MHC) class II molecules and T-cell receptors resulting in extremely strong immunostimulatory properties. Staphylococcus aureus enterotoxin A (SEA) belongs to a subgroup of the staphylococcal superantigens that utilizes Zn2+ in the high affinity interaction with MHC class II molecules. A high affinity metal binding site was described previously in SEA co-crystallized with Cd2+ in which the metal ion was octahedrally co-ordinated, involving the N-terminal serine. We have now co-crystallized SEA with its native co-factor Zn2+ and determined its crystal structure at 2.7 Å resolution. As expected for a Zn2+ ion, the co-ordination was found to be tetrahedral. Three of the ligands are located on the SEA surface on a C-terminal domain β-sheet, while the fourth varies with the conditions. Further analysis of the zinc binding event was performed using titration microcalorimetry, which showed that SEA binds Zn2+ with an affinity of KD = 0.3 μM in an entropy driven process. The differential Zn2+ co-ordination observed here has implications for the mechanism of the SEA-MHC class II interaction.