PRESENTATION OF ANTIGEN, INTRODUCED INTO THE CELLS BY STIMULATION OF MACROPINOCYTOSIS

Abstract: The invention provides a method for the introduction of a substance into living cells via ingestion by macropinocytosis. Macropinocytosis can be stimulated using various agents, and the substance to be introduced may be directed to specific cellular targets by being linked with a molecular signal. The invention may be used to introduce vaccines. In particular, proteins taken up by macropinocytosis can gain access to the cytosol and therefore into the conventional Class 1 MHC pathway.

Description: PRESENTATION OF ANTIGEN, INTRODUCED INTO THE CELLS BY STIMULATION OF MACROPINOCYTOSIS This invention relates to a method for introducing a substance into living cells. The invention particularly relates to the phenomenon of macropinocytosis which occurs in mammalian cells, and the exploitation of this phenomenon in the method. Macropinocytosis is a form of endocytosis which is clathrin- and receptor-independent and allows the cell to ingest large amounts of matter (eg liquids, proteins etc) in a non-specific manner. During macropinocytosis, the outer membrane of the cell ruffles and envelops the matter to be ingested and large vesicles detach from the cell membrane to form macropinosomes, which are much larger endocytic vesicles (2-5μm) relative to those formed by clathrin- mediated endocytosis (O.lμm). Macropinosomes are heterogenous in size (Swanson, 1989; Hewlett et al., 1994) and interact with lysosomes in macrophages (Racoosin & Swanson, 1993) but in human A431 epithelial cells they comprise a distinct endosome population refractory to fusion with other endosomes (Hewlett et al. , 1994). According to the present invention there is provided a method for the introduction of a substance into living cells, the method comprising providing the substance at a location adjacent to the cell membrane, and allowing ingestion of the substance into the cell by macropinocytosis. The macropinocytosis may be accompanied or preceded by ruffling of the cell membrane. Agents which stimulate macropinocytosis and/or ruffling can be useful but not essential in the method to stimulate ingestion of the substance by the cell. Useful stimulants include phorbol esters such as PMA, or MCSF (macrophage colony stimulating factor) , and growth factors such as hepatocyte growth factor/scatter factor or epidermal growth factor etc. Typically, in certain cell types appropriate growth factors will be more useful than others. Other stimulants may include bacterial or yeast cell wall components etc, activation of the small GTP binding protein rac, and other GTP binding proteins. However, in some cell types (eg cultured human dendritic cells) macropinocytosis can operate constitutively (eg without exogenous stimulation) . Macropinocytosis, but not other endocytic pathways, is blocked by the Na+/H+ channel inhibitor amiloride or its analogues. An important preferred step is to link the substance to be introduced with a molecular signal (such as a signal peptide) to direct the matter to a specific cellular target. This can be of particular benefit in the introduction of factors which can modulate gene expression (for example), which can be directed to the nucleus by the signal peptide -CGGGPKKKRKVED. The use of the macropinocytosis pathway for ingestion of the substance allows the non-specific introduction of the substance into the cell, and particularly into the cytosol, since macropinosomes can release their contents into the cytosol, and thence, via molecular signals to other organelles (eg the nucleus) . It may also be possible for macropinosomes to fuse with particular organelles, particularly endocytic organelles. The substance can comprise macromolecules such as proteins, sugars, peptides, nucleic acids (RNA or DNA) , gene transcription factors, or any combination or complex of such macromolecules (eg such as nucleic acid/protein complexes) . DNA vaccines and agents for gene therapy are two possible substances which can be delivered (preferably via the cytosol) to the nucleus or to other organelles as necessary. Because the macropinosomes are large (up to 5μm diameter) , they can ingest large volumes of matter; it is therefore potentially feasible for the present invention to allow the introduction of relatively large particles into the cytosol such as liposomes and polymer aggregates, bacteria and yeast etc. It is also feasible for the method of the invention to enhance delivery into target cells of other cells eg tumour cells and/or their proteins or fragments (eg apoptotic fragments) . Mention may be made in this regard of gp96, a binding protein purified from tumour cells which may act as a cancer vaccine; gp96 may provide a store of peptides for class I MHC to bind including tumour specific peptides. The method can be used to enhance the uptake and/or effectiveness of existing delivery vehicles such as liposomes and polymers etc. Stimulators of ruffling and macropinocytosis can be incorporated into such vehicles. The invention can be used to introduce conventional or tumour antigens into professional antigen presenting cells (eg macrophages or dendritic cells) which may then trigger lymphocytes eg CD8 T-cells, which have anti-viral and anti-tumour characteristics when stimulated. Stimulation of these cells is particularly difficult because stimulating antigens must interact with cytosolic components (such as the cytosolic proteosome complex and TAP transporter) . Previously, live attenuated viral vectors have been used to ensure synthesis of viral proteins in the cells. It has been shown that professional antigen presenting cells must be recruited to trigger CD8 T-cell responses, and the method can be used to deliver antigens into the cytosol of professional antigen presentation cells (eg macrophages and dendritic cells) to trigger the activation of the CD-8 T-cells. In such cases the immunogen can be coupled or admixed with an agent to stimulate macropinocytosis. The delivery of antigens to the cytosol by the method of the present invention allows enhanced antigen loading of the cells. This loading can be achieved in vivo or in vitro. For example, macrophages, dendritic cells or other professional antigen presenting cells can be cultured and stimulated in vitro and thereafter can be returned to the body of the donor. Alternatively, the cells can be stimulated direct .in vivo. Other cell types to which antigens or stimulants can be presented more effectively by the method of the present invention include other types of leucocytes, macrophages, neutrophils, dendritic cells etc. T lymphocytes are central to virtually all immune (and autoimmune) responses and their activity is controlled by recognition of either class I of class II MHC (Major Histocompatibility Complex) molecules. These are two classes of MHC molecules expressed on the cell surface, class I and class II, which are recognised by two classes of T lymphocyte, CD8 and CD4 T cells respectively. CD8 T cells are cytotoxic and the outcome of MHC recognition is killing of the target cell. These responses are important in anti-viral and anti-tumour immunity and in the clearance of certain intracellular parasitic infections. In contrast CD4 T cells provide a helper/inducer function which allows cells expressing appropriate class II MHC molecules, such as B lymphocytes and macrophages, to differentiate and in the case of B lymphocytes, become antibody secreting cells. CD4 T cells are therefore crucial, for example, to antibody responses. A receptor on the T cells recognises the MHC molecule plus a short peptide (8-10 amino acids on class I; -12-25 on class II) derived from the foreign antigen (antigen processing) . These peptides are tightly bound in a groove or channel on the top of the MHC molecule and the T cell receptor makes contact with both the MHC molecule and the peptide (antigen presentation) . MHC molecules capture peptides during their assembly and transport to the cell surface and are scrutinised by the T cell population for the appearance of any newly appearing foreign peptides. The peptides displayed on class I and class II MHC molecules come from proteins located in very different cellular compartments. Class I molecules capture peptides derived from cytosolic (and nuclear) proteins whereas class II molecules capture peptides derived from extracellular proteins (ie those taken up by endocytosis or phagocytosis) . In effect this means that for loading of class I MHC and triggering of CD8 responses the antigen must be either synthesised in the cytosol or somehow gain access to the cytosol from outside. Since there is no general mechanism for proteins to breach this topological barrier one cannot generally vaccinate for CD8 T cell responses without using live attenuated virus or specialised antigen delivery systems which can cross the membrane barrier into the cytosol. A further restriction is that only certain cell types can trigger CD8 T cell responses. These cells are typically macrophages and another bone marrow derived cell, the dendritic cell. Thus a general method of loading the cytosol of these cells would permit access of 'exogenous' antigens to the class I MHC loading pathway. Since macropinocytosis is very active in both these cell types and can be further increased by a variety of stimuli, this topological barrier can be breached and under these exceptional conditions, class I MHC molecules can be loaded and CD8 T cells activated. Macropinocytosis can lead to effective targeting into the class II MHC pathway. Since CD4 T cell 'help' is thought to be necessary for triggering CD8 T cell responses, peptides recognisable by both T cell types may be presented on the same antigen presenting cell following uptake of exogenous antigen by macropinocytosis providing particularly favourable conditions for CD8 T cell priming. The conventional view of antigen presentation to T cells is that endogenous proteins are presented on class I MHC molecules and exogenous proteins on class II MHC molecules (Morrison et al., 1986; Bevan, 1987; Townsend & Bodmer, 1989; Germain & Marguiles, 1993) . However, this division is not absolute and peptides from endogenous cytosolic proteins may appear on class II MHC molecules (Jarque ada et al. , 1990; Dodi et al., 1994) and peptides from exogenous proteins on class I MHC molecules. CD8 T cell responses can be elicited in vivo in the absence of de novo antigen synthesis within host antigen presenting cells. These responses have been observed following immunisation with purified proteins (Wraith et al., 1987: Staerz et al., 1987; Bachmann et al., 1994; Schrimbeck et al., 1994; Blum- Tirouvanzia et al., 1994; Ke et al., 1995) and in 'cross-priming' experiments where CD8 T cell responses to antigens carried uniquely by immunising donor cells are restricted to MHC molecules expressed only on recipient cells (Bevan, 1976; Godding and Edwards, 1980; Fink et al., 1983; Huang et al, 1994) . Based on these findings it has been suggested that a pathway must exist for the processing and presentation of exogenous material on class I MHC molecules (Bevan, 1987; Townsend & Bodmer, 1989). However, it has been difficult to analyze this unusual mode of class I peptide loading in vitro where exogenous proteins are usually presented on class II MHC and not on class I MHC molecules (Morrison et al. , 1986). Rock and colleagues have demonstrated class I MHC-restricted presentation in vitro of exogenous soluble antigen (Rock et al., 1990) and much more efficient presentation of antigen conjugated to latex beads (Kovacsovics-Bankowski et al., 1993). However, the mechanistic basis for class I MHC presentation of phagocytosed particulate antigens is somewhat unclear since different studies, both using latex bead associated antigen, report a brefeldin A-insensitive pathway in one case (Harding and Song, 1994) but brefeldin A-sensitive presentation in another (Kovacsovics-Bankowski & Rock, 1995) . Brefeldin insensitive presentation of bacterial antigen (Pfeiffer et al., 1993) and heat-inactivated virus preparations in some cell types (Liu et al. , 1995) have also been described. Thus the question of how exogenous physiological antigens, both particulate and non- particulate, might access the class I MHC pathway following endocytosis is not clearly resolved. The present invention will now be illustrated by way of the following examples with reference to the accompanying figures wherein:- Figure 1 illustrates release of solute markers into the cytosol following induction of macropinocytosis. (a) A431 cells incubated with HRP in the presence (A,C,D) or absence (B) of EGF for 10 minutes, then fixed and peroxidase cytochemistry performed immediately (A) or following a 90 minutes chase (B,C,D) . Macropinosomes, heterogeneous in size, can be seen close to the ruffling margins of a group of cells (A) . (b) Quantitation, after different chase times of cells showing cytosolic HRP reaction product. A cell was scored if it excluded trypan blue and the brown HRP precipitate was excluded from the nuclear region. In all cases a minimum of 500 cells were counted. Scale bars, Fig. la = 20μm (A,D); 50μm (B,C) . A431 cells were grown on sterile 13mm coverslips, then pulsed with 10 mg/ml HRP in the presence or absence of 300ng/ml EGF as described previously (West et al, 1989; Hewlett et al, 1994) and where indicated, chased at 37°C. After fixation, HRP cytochemistry was preformed with diaminobenzidine (0.5mg/ml) and H 2 0 2 (0.02%). Figure 2 illustrates bone marrow macrophages (BMM) pulsed with FITC-dextran in the presence of MCSF. Phase contrast (A,C) and confocal fluorescence (FITC) fields of the same cells showing macropinosomes (B,D) and less frequently, cells showing cytosolic fluorescence (arrow in C&D) . BMM were grown as described (Racoosin & Swanson, 1989) detached from the plastic and replated at 1.5 x 10 5 cells/ml onto sterile 24mm coverslips. Following pulsing with FITC-dextran (5mg/ml) for 10 minutes in the presence of 3000U/ml MCSF the cells were washed in PBS/BSA, then mounted in 0.4% trypan blue in HBSS/BSA in a Dvorak-Stotler perfusion chamber for viewing in Biorad MRC 500 laser confocal microscope at 30-37°C. Figure 3 illustrates the effects of PMA and amiloride on macropinocytosis and class 1 presentation of exogenous antigens. BMM untreated (D) or treated (■) with 30ng/ml PMA to induce macropinocytosis 45 minutes prior to pulsing with either ovalbumin (a,b,c), ovalbumin 257-264 (e) or FITC-dextran (d) . Time- dependent presentation of native ovalbumin by ovalbumin-pulsed cells (a) . Effect on ovalbumin (b,c) or ovalbumin 257-264 (e) presentation by amiloride (b) or dimethyl amiloride (c,e) . Effect of dimethyl amiloride on FITC-dextran uptake into BMM (d) . BMM were replated at 3 x 10 5 cells/well in 24-well plates 6-18 hours and PMA added where indicated (■) 45 minutes prior to pulsing with batch tested (see Experimental procedures) ovalbumin (lOmg/ml) or ovalbumin 257-264 (O.lμM) or FITC-dextran (lOmg/ml) . For presentation studies BMM were washed extensively with PBS/BSA following ovalbumin pulsing then incubated for 2 hours at 37°C. B3Z cells were overlaid at a density of 4 x 10 5 cells/well and incubated overnight at 37°C, then harvested by vigorous pipetting, β- galactosidase activity was assayed using X-Gal as described (Sanderson and Shastri, 1994; Kartunnen et al, 1992). In (a) cells were fixed with 1% paraformaldehyde for 30 minutes and quenched with 0.2M glycine in PBS, washed and then incubated with B3Z indicator cells. The percentage of activated, blue B3Z cells was determined by cell counting. At least 1500-3000 total cells were counted. Experiments were performed in triplicate and the results presented as means ±S.D. In (d) PMA treated cells were washed extensively following exposure to FITC-dextran (5mg/ml) then lysed in 0.2% Triton-X 100 and icrofuged to remove aggregates. FITC content was measured in a Perkin-El er fluorimeter (excitation 490nm, emission 520nm, 2.5nm bandpass). Controls without PMA or amiloride received equivalent amounts of DMSO carrier. Figure 4 illustrates the effects of gelonin, brefeldin A, LLnL, LLM and chloroquine on presentation of acropinocytosed ovalbumin on class 1 MHC molecules assessed using B3Z indicator cells. (a) BMM pulsed with ovalbumin in the presence or absence of gelonin. (b) BMM pulsed with ovalbumin and chased in the presence or absence of brefeldin A for 6 hours. B3Z cells were overlaid on either fixed cells or live cells washed free of brefeldin A. Ovalbumin pulsing of BMM in the presence or absence of LLnL or LLM (c) (or chloroquine (d) . BMM were pretreated with PMA 45 minutes prior to ovalbumin pulsing. Times of addition of inhibitors prior to ovalbumin pulsing were: Gelonin (lμM) , 20 minutes; LLnL or LLM (N-acetyl-L-leucinyl-L-leucinal-L- norleucinal and N-acetyle-L-leucinyl-L-leucinal- methional respectively; both 50μM) or chloroquine (lOOμM) all at 60 minutes. Brefeldin A (5μg/ml) was added after ovalbumin pulsing and was replenished in the culture medium at 120 minute intervals. Cells were chased for 6 hours following ovalbumin pulsing and then overlaid with B3Z cells either with (c,d and where indicated in b) or without a) prior fixation. Following an overnight incubation with B3Z cells, T cell triggering was measured as described and Figure 3 legend. Samples without drugs contained equivalent amounts of DMSO or ethanol carrier. Figure 5 diagrammatically illustrates delivery of antigen by macropinocytosis into the class 1 MHC pathway. Figure 6 illustrates TAP dependent presentation of macropinocytosed antigen. Figure 7 illustrates PMA stimulation of processing by dendritic cells and macrophages. Figure 8 illustrates PMA stimulation of class 1 MHC restricted presentation of OVA as assayed by CPRG. Figure 9 illustrates macromolecules can target the cell nucleus. Experimental Procedures Reagents EGF was purchased from AMS Biotechnology (Witney, oxon, U.K.) . Cell culture media and G418 were from GIBCO BRL (Paisley, Scotland) . 100mm Lab-Tek tissue culture dishes were from Nunclon (Roskilde, Denmark) and all other tissue plastics were from Costar (Bucks, U.K.). Brefeldin A and hygromycin B were purchased from Boehringer Mannheim (East Sussex, U.K.). Foetal calf serum was from Advanced Protein Products (West Bromwich, U.K.). 5-Bromo-4-chloro-3-indolyl-/3-D- galactoside (X-Gal) was from Calbiochem-Novabiochem (Nottingham, U.K.). Glass cover slips and slides were from BDH Chemicals Ltd (Poole, U.K.). All other reagents were from Sigma Immunochemicals (Poole, U.K.). Cell Culture A431 cells (a generous gift from Dr Paul Smith, University College, London) were maintained in DMEM containing heat denatured FCS (10%), kanamycin (lOOU/ml) and glutamine (2mM) . B3Z cells were maintained in RPMI 1640/10%FCS supplemented with sodium pyruvate (ImM) , non-essential amino acids (ImM), glutamine (2mM) , kanamycin (lOOU/ml) , hygromycin B (400U/ml) and geneticin (G418; 1 mg/ml). Bone marrow macrophages (BMM) were obtained from C57BL/6J female mice, and expanded in L-cell supernatant as described by Racoosin and Swanson (1989) . BMM were cultured in 30% L-cell supernatant for 5-7 days. The macrophages were detached from dishes by washing briefly with ice- cold PBS, incubation with 10ml PBS containing lOmM EDTA, 20% FCS at 37°C for 10 minutes and agitating vigorously with an automatic pipettor. For assays BMM were cultured at 10 5 -2 x 10 5 per well in 24-well plates coated with poly-L-lysine or onto sterile 24mm circular glass cover slips for microscopy. Greater than 95% of the cells were macrophages as determined by immunofluoresence staining with a monoclonal antibody (Ml/70.15.1, Serotec, Oxford, U.K.) to the macrophage cell surface marker Mac-1. Uptake of fluid phase markers Uptake of the fluid phase markers horseradish peroxidase (HRP) and fluorescein isothiocyanate (FITC) conjugated dextran was detected by microscopy. Exposure to these solute markers was performed as previously described (West et al 1989 and Hewlett et all 1994, respectively) . Prior to use the HRP and FITC-dextran solutions used were passed through a 0.22μm filter. A431 cells were grown on 13mm diameter glass cover slips, thickness no.2. Cells were incubated with HRP for 10 minutes, washed, then chased in culture medium in the absence of HRP for the times indicated before rinsing in PBS, exposure to 0.4% trypan blue in PBS for 2 minutes and then fixation in 0.5% glutaraldehyde. HRP activity was developed with 0.5mg/ml diaminobenzidine (DAB) and 0.01% hydrogen peroxide in PBS. BMM were grown on 24mm cover slips and exposed to the solute marker FITC-dextran (5mg/ml) in a similar manner, in the presence or absence of either 30ng/ml PMA or 3000U/ml M-CSF. Cover slips were mounted in a Dvorak-Stotler perfusion chamber in PBS/0.4% trypan blue and viewed at 30-37°C on a Nikon Microphot-SA attached to a confocal laser scanning microscope (MRC- 600 series, Bio-Rad Laboratories, Cambridge, U.K.) . Fluid phase pinocytosis was quantitated by measuring the uptake of the solute marker FITC-Dextran by fluorimetry, essentially as described by Racoosin and Swanson, 1989. Quantitation of FITC-Dextran uptake, after a two hour exposure, was performed by excitation at 488nm (2.5nM bandpass) and measurement of emission at 520nm (2.5nM bandpass). Antiαen Uptake and Presentation Presentation of ovalbumin 257-264 on K b (Moore et al, 1988) was detected using the T cell hybridoma B3Z which carries a 0-galactosidase construct driven by NFAT elements from the IL-2 promoter (Sanderson and Shastri, 1994; Kartunnen et al, 1992). Ovalbumin (Lome Labs; Batches 30C571N or 34M877) gave a background of T cell triggering upon incubation with fixed antigen presenting cells of <0.1% activated B3Z cells. BMM, obtained as described above, were pulsed with lOmg/ml Ovalbumin dissolved in serum free DMEM for 30 minutes and fixed with 1% paraformaldehyde for 30 minutes. Excess fixative was quenched with 0.2M glycine in PBS and the BMM were overlaid with B3Z cells overnight. The B3Z cells were assayed for β-galactosidase production with X-Gal as described by Sanderson et al., 1994. Activation of the B3Z cells was quantitated by counting the number of blue (LacZ positive cells) on a Neubauer counting chamber and expressing the result as a percentage of the total number of cells present. In all cases a minimum of 1000 cells were counted. For antigen presentation assays BMM in a 24-well plate were pulsed with 250μl pre-warmed OVA (lOmg/ml) in DMEM with or without 30ng/ml PMA for a period of ten minutes. The cells were washed 3 times with PBS/BSA (1 mg/ml) . Following loading the cells were chased for 2 hours in complete DMEM before overlaying with 3.5 x 10 5 B3Z cells in complete RPMI and incubating overnight. To remove the B3Z cells the plates were spun at 400rpm for 3 minutes and the majority of the medium then removed. Vigorous pippetting of the remainder of the medium removed the majority of B3Z cells and incubation at room temperature in trypsin/EDTA dislodged the remainder. After washing twice LacZ activity within the cells was developed as described above. Where indicated the following inhibitors were included in the antigen presentation assays. Amiloride and 5- (N,N-Dimethyl) -Amiloride (DMA) were stored as stock solutions of 0.6M and lOmM, respectively, in DMSO, at 0°C, and diluted in culture media for use. Gelonin was used at a final concentration of lμM after dilution, in culture medium, from a stock solution, 20μM in DMSO. BMM were incubated with amiloride and DMA and gelonin for 10 minutes and 20 minutes, respectively prior to OVA pulsing and during the OVA pulse. Brefeldin A was prepared at 5mg/ml in DMSO and was used at 5ug/ml in complete DMEM for six hours. As Brefeldin A has a short lifetime in culture the medium containing it was replaced every two hours. Following a six hour chase cells were either fixed with paraformaldehyde, as outlined above, or overlaid with B3Z and incubated overnight as indicated. The peptide aldehydes LLnL and LLM (50μM) and the lsyosomotropic amine chloroquine (lOOμM) were pre-incubated for the same period with BMM as PMA, and the cells were chased in the presence of these inhibitors for a further six hours before fixation. In all cases control cultures were incubated in DMSO at a concentration equivalent to that in the inhibitor preparation. Example l: Detection of macropinocytosed markers in the cytosol. In human A431 cells stimulated with EGF in the presence of horseradish peroxidase (HRP) , macropinosomes of various sizes can be observed in the cytosol, frequently in close proximity to the cell margins where ruffling activity is maximal (Fig. la, panel A) (West et al., 1989; Hewlett et al., 1994) . Although most macropinocytosed tracer is recycled out of these cells during a chase period at 37°C we detected HRP activity in the cytosol in a proportion of cells (Fig. la, panels C&D) . These cells were viable since HRP was excluded from the nucleus (Fig. 1, panels C&D) and the cells did not take up trypan blue (not shown) . Cytosolic HRP was seen only in EGF-stimulated cells (Fig. la, compare panels B & C) which also contained intact macropinosomes and were therefore alive during the HRP pulse. Most importantly, HRP only appeared in the cytosol following a chase period, ie after removal of exogenous HRP, demonstrating a time dependent release into the cytosol. Approximately 9% of cells were visibly loaded with cytosolic HRP following a 90 minute chase period (Fig. la, C&D and Fig. lb) . This surprising observation raised the possibility that macropinocytosis might provide a pathway for the presentation of exogenous antigens on class I MHC molecules via the cytosol. We tested this hypothesis using mouse bone marrow macrophages (BMM) which constitute a physiologically relevant cell type in the context of antigen presentation and CTL priming. Mouse BMM were stimulated with either MCSF or phorbol ester (which stimulate membrane ruffling and macropinocytosis) in the presence of FITC-dextran. HRP is less suitable as a fluid phase endocytic marker in BMM since it stimulates its own uptake (Swanson et al. , 1995) . As in A431 cells, macropinosomes in BMM were heterogenous in size (Fig. 2c, panels A & B) and their formation could be blocked by amiloride (which blocks membrane ruffling and macropinocytosis) or its analogues (data not shown and Fig. 3c, see below) . In addition we observed FITC-dextran release into the cytosol of some living cells (Fig. 2c, panels C & D). However, since cytosolic release was again seen only in a minority of cells it seemed that although BMM would not be efficiently lysed in a CTL assay following induction of this pathway they might nonetheless be able to trigger T cell activation. We therefore utilised the Lac Z-inducible T cell hybridoma B32 (Karttunen et al., 1992), specific for the ovalbumin epitope 257-264 presented on the murine class I MHC molecule H-2K (Moore et al., 1988) . B3Z activation is detectable when the frequency of presenting cells is as low as 0.1-0.01% (Karttunen et al., 1992; Sanderson and Shastri, 1994) . Example 2: Macropinocytosed antigen is loaded onto class I MHC molecules. BMM were pulsed with soluble ovalbumin for 10 minutes under conditions which stimulate or inhibit macropinocytosis. The cells were then incubated without ovalbumin for different times, fixed and overlaid with B3Z T cells. Presentation of the ovalbumin 257-264 epitope was assessed by scoring the number of activated (blue) B3Z T cells. Exposure of BMM to ovalbumin in the presence of PMA gave a time- dependent increase in triggering of the B3Z T cells (Fig. 3a) . Amiloride added during the antigen pulse markedly reduced subsequent T cells triggering (Fig. 3B) . Thus, presentation of exogenous ovalbumin was enhanced or blocked, respectively, by stimulators and inhibitors of macropinocytosis. To eliminate the possibility that PMA and amiloride were modulating presentation of ovalbumin via generalised effects, for example, on the level of class I MHC or adhesion molecules, we compared the effect of PMA and the more potent amiloride analogue, dimethyl amiloride (DMA) on ovalbumin presentation versus presentation of a synthetic ovalbumin 257-264 peptide (SIINFEKL) recognised by B3Z cells. The DMA concentrations that blocked PMA-sti ulated macropinocytosis of FITC-dextran (Fig. 3d) also inhibited PMA-stimulated and constitutive presentation of intact ovalbumin (Fig 3C) . In contrast, even high DMA concentrations had no detectable effect on peptide presentation. Moreover, in contrast to ovalbumin presentation peptide presentation was, if anything, slightly inhibited by PMA (Fig 3e) . The inhibitory effect of DMA on class I presentation correlates strictly with its parallel effects on macropinocytosis since it inhibited ovalbumin presentation only when it was included during the antigen pulse and not when it was present for the same length of time either before or after pulsing (Table 1) . Table 1. Dimethyl amiloride inhibits presentation during but not before or after OVA pulse. Window of DMA Exposure None Pre- Co- Post- % Lac-Z +ve cells +PMA 2.73 1.78 0.39 2.42 -PMA 0.39 n.t n.t n.t BMM were stimulated with PMA 45 minutes prior to OVA pulsing. Dimethyl amiloride (DMA; lOOμM) was included in separate incubations during different 20 minute periods and then removed. Exposure to DMA was either at the start of PMA stimulation (Pre-) , 10 minutes prior to and during the 10 minutes OVA pulse (Co-) or after removal of OVA (Post-) . Following a 2 hour chase, cells were overlaid with B3Z and presentation was quantitated as described in Figure 3 legend and in Methods, n.t., not tested. BMM exhibit a significant level of constitutive ruffling and macropinocytosis which presumably accounts for the DMA-inhibitable presentation of ovalbumin seen in the absence of PMA (eg figure 3c) . Thus PMA and DMA modulate antigen presentation via their effects on ruffling and macropinocytosis and not by modulating cell surface expression of class I MHC or accessory molecules. Example 3 : Unconventional access to the conventional class I MHC pathway. Release of exogenous markers into the cytosol following macropinocytosis seemed likely to account for the increased expression of exogenous ovalbumin on class I MHC molecules. However, other studies have suggested that exogenous antigens may be loaded onto mature class I MHC molecules (Pfeifer et al., 1993; Harding and Sond, 1994; Liu et al., 1995). To assess whether newly synthesised class I molecules and a functional secretory pathway are required for presentation of macropinocytosed ovalbumin we tested the effects of gelonin, a protein synthesis inhibitor (Stirpe et al., 1980) , and brefeldin A, which blocks secretory protein traffic and class I MHC antigen presentation (Nuchtern et al. , 1989; Yewdell and Bennink, 1989) . Gelonin added during the antigen pulse inhibited ovalbumin presentation (Fig. 4a) but not presentation of the synthetic peptide ovalbumin 257-26 (not shown), indicating that newly synthesised class I MHC molecules are required for presentation of macropinocytosed ovalbumin. Since gelonin is impermeant to intact cells (Stirpe et al., 1980; Rock et al., 1993) these data provide independent evidence that macropinocytosis provides access of soluble proteins to the cytosol. When ovalbumin-pulsed macrophages were subsequently incubated with brefeldin A, triggering of B3Z cells was strongly inhibited (Fig. 4b) . However, following a period of recovery in the absence of brefeldin A and without further exposure to ovalbumin, the macrophages became competent to activate the B3Z cells (Fig. 4b) , demonstrating that the effect of brefeldin A is reversible and that presentation of macropinocytosed ovalbumin depends on a functional secretory pathway. We next assessed whether, prior to class I MHC loading, processing of ovalbumin occurred within macropinosomal compartments or in the cytosol via the action of the proteasome complex. Presentation of exogenous ovalbumin to B3Z cells was blocked by the peptide aldehyde inhibitor LLnL (Fig. 4c) . Since LLNL and other peptide aldehydes also inhibit some classes of lysosomal protease (Sasaki et al, 1990) we also tested the analogue LLM which has a selectively reduced activity against the proteasome and in contrast to LLnL, had no effect on presentation of ovalbumin introduced into the cytosol by electroporation (Rock et al., 1994) . As shown in Fig. 4C, LLM also had no effect on presentation of macropinocytosed ovalbumin (Fig. 4C) . Similarly, chloroquine a pleiotropic inhibitor of acid-pH dependent protease did not inhibit ovalbumin presentation and in fact showed a reproducible stimulation indicating that macropinosomal antigen processing is unnecessary and might actually reduce the efficiency of class I MHC presentation of exogenous antigens (Fig. 3d). Thus, cytosolic rather than macropinosomal processing of ovalbumin was necessary for class 1 presentation. Example 4: Presentation of exogenous soluble antigen is TAP-dependent Macrophages from normal mice or from TAP gene deleted mice were exposed to exogenous ovalbumin in the presence or absence of PMA to stimulate macropinocytosis. Presentation on class I MHC was scored using the T cell B3Z. As a control both sets of cells were offered the peptide ovalbumin 257-264 which can bind directly to cell surface class I MHC, TAP deleted cells are seen to be equally effective under these conditions and therefore express the appropriate class I MHC molecules. The results in Fig 6 show that the so called TAP transporter proteins located in the endoplasmic reticulum are necessary for presentation of exogenous soluble antigen on class I MHC molecules. This was established using macrophages established from bone marrow of mice in which the TAP genes were deleted by homologous recombination. This observation is not unexpected given the data we already have but underlines the fact that the conventional class I loading pathway is being utilised following transfer of antigen into the cytosol. Example 5: Class I MHC presentation of exogenous soluble antigen bv dendritic cells Much evidence suggests that the most potent cells for inducing T cell responses are dendritic cells. These are derived from bone marrow and can be isolated from blood and from peripheral tissue. Following protocols for the generation of these cells from bone marrow using granulocyte/macrophage colony stimulating factor (GMCSF) we have now shown that these cells are more potent than macrophages at presenting an exogenous antigen on class I MHC (see figs 7 and 8) . As shown in fig 3, dendritic cells (DC) are more potent even without stimulation of macropinocytosis by phorbol ester (PMA) then macrophages under PMA stimulation. However, presentation of antigen to CD8 T cells is boosted further by stimulation of the cells with PMA. Preliminary experiments suggest that many cells in the dendritic cell population can transfer the horseradish peroxidase marker into the cytosol following macropinocytosis. With reference to Figs 7 and 8, cells were exposed to different concentrations of the extracellular (exogenous) antigen ovalbumin. Triggering of the CD8 T cell clone B3Z was measured either by quantitation of the number of T cells expressing 0-galactosidase or by measuring the total (3-galactosidase activity in the T cell population using the substrate chlorphenol red β- galactoside (CPRG) and detection at OD590nm (Fig 7) . Example 6: Nuclear localisation of macropinocytosed substances. A431 cells were stimulated to macropinocytose with 50ng/ml EGF in the presence of ~lmg/ml horse radish peroxidase (HRP) conjugated to the nuclear localisation signal CGGGPKKKRKVED. After 10 min both the growth factor and the HRP were removed and the cells were incubated for 45 min before being fixed. The peroxidase activity was localised with the substrates DAB and H 2 0 2 . Shortly after stimulation the cells showed peroxidase present in the macropinosomes or in the cytosol but following additional incubation the peroxidase is clearly seen to be targetted to the nucleus. Example 7: Delivery of marker by constitutive macropinocytosis Mouse dendritic cells were expanded from mouse bone marrow cultures in GMCSF (granulocyte macrophage colony stimulating factor) for 7 days. The cells were incubated with 2mg/ml HRP for 20min. At the end of this period the cells were washed, fixed and the HRP activity was localised as described above. Considerable constitutive macropinocytosis occurs in these cells and that the macropinosomes appear as brown spherical intracellular structures. Some cells show only these structures but most show in addition the clear presence of HRP in the cytosol. Macropinosome to cytosol transfer of the marker has occurred in a high proportion of the cells. This suggests that proteins with specific regulatory nuclear functions such as transcription factors could be taken up by cells in similar fashion. Example 8: Macromolecules taken up via the ruffling/macropinocytosis response can target to the cell nucleus Horse radish peroxidase (HRP) was conjugated to the peptide CGGGPKKKRKVED which contains the nuclear targeting signal (NLS) from the SV40 T antigen (Kalderon et al, 1984) . A431 cells were stimulated with lOOng/ml epidermal growth factor in the presence of -200μg/ml HRP-NLS for 10 minutes. The cells were washed and then incubated for 45 minutes at 37°C in the absence of EGF or HRP. After fixation peroxidase cytochemistry was performed as described (West et al, 1989) with diaminobenzidine and hydrogen peroxide as substrates. In contrast to cells incubated under the same conditions with HRP (Norbury et al, 1995) where nuclear localization was notably absent, cells incubated with HRP-NLS show clear targeting of the enzyme to the nucleus of living cells (Figure 9) . Constitutive macropinocytosis allows TAP-dependent major histocompatibility compex class I presentation of exogenous soluble antigen by bone marrow-derived dendritic cells Dendritic cells expanded from mouse bone marrow (BMDC) with granulocyte/macrophage colony stimulating factor have potent T cell stimulatory properties both in vitro and in vivo . An unresolved question has concerned the capacity of BMDC to present exogenous antigen on major histocompatibility complex (MHC) class I molecules, an unconventional mode of MHC class I loading for which there is now considerable evidence, particularly in macrophages. Here, we show that BMDC exhibit high levels of macropinocytosis drive by constitutive membrane ruffling activity. Up to one-third of actively ruffling and macropinocytosing BMDC transferred pinocytosed horseradish peroxidase into the cytosol following a 15-min pulse, suggesting that they might be capable of presenting exogenous soluble antigen on MHC class I molecules. We show that BMDC presented exogenous ovalbumin to a T cell hybridoma more effectively, more rapidly, and at lower exogenous antigen concentrations than BM macrophages on a cell- for-cell basis. Presentation was TAP-dependent, brefeldin A-sensitive, and blocked by inhibitors of proteasomal processing, demonstrating use of the classical MHC class I pathway. Although effective presentation of exogenous antigen by BMDC occurred in the absence of agents which stimulate macropinocytosis, treatment with phorbol myristate acetate (PMA) enhanced both pinocytosis and MHC class I presentation by BMDC. Finally, PMA-stimulated BMDC exposed to exogenous ovalbumin in vitro were able to prime an antigen- specific cytotoxic T lymphocyte response following adoptive transfer in vivo . BMDC can also process and present exogenous soluble protein antigen. However, whereas presentation by bone marrow-derived macrophages required stimulation with phorbol esters or growth factors, BMDC present exogenous antigen constitutively on their MHC class I molecules following antigen capture via constitutive macropinocytosis. Delivery of an exogenous tracer to the cytosol could be demonstrated in approximately 30% of cells. Using DC expanded from TAP-/- bone marrow, we show that presentation of exogenous antigen via this pathway is TAP dependent. BMDC presented exogenous antigen on MHC class I considerably more efficiently than bone marrow-derived macrophages from parallel cultures, and following brief exposure to exogenous antigen in vitro , could prime CTL responses in vivo . Figure 10 illustrates constitutive membrane ruffling and macropinocytosis in BMDC. Cells in slide flasks were viewed at 37°C as described in Section 2.4. In (a) , the cell in the centre of each panel shows three different episodes of ruffling and macropinosome formation over an 8-min period. In addition, it is moving from top right to bottom left over the same period. Ruffling in a characteristic circular morphology and the formation of two macropinosomes (0- 160 s) is then followed by ruffling and macropinosome formation at a second site (arrowed; 240-300 s) . The cell appears to contact a second cell and engages in a third episode of ruffling and formation of several large macropinosomes (300-480 s) . Once formed macropinosomes move in towards the nucleus. Figure 11 illustrates constitutive acrophinocytosis in BMDC can load exogenous tracers into the cytosol. (a) Phase-contrast image of BMDC exposed for 15 min to HRP. All cells show peroxidase-loaded macropinosomes and several show evidence of cytosolic HRP revealed by DAB cytochemistry. The central well-spread cell illustrates best cytosolic, but not nuclear HRP staining as well as several intact macropinosomes. In (b) , the proportion of cells exhibiting labelled macropinosomes (D) or cytosolic loading (■) after different times of pulsing with HRP is shown. For all points, 500 cells were counted, and cells were only scored as cytosolically labelled if the nucleus remained free of HRP. Figure 12 illustrates quantitation of BMDC pinocytic activity. (a) BMDC were incubated with 1 mg/ml HRP for different time periods in the presence (■) or absence (D) of 1 mg/ml yeast mannan, and in (b) , in the presence (0) or absence (•) of 30 ng/ml PMA. At the end of each incubation period, the cells were washed extensively and HRP activity measured in cell lysates and converted to apparent volumes using an HRP standard curve. Readings shown are means of a minimum of three independent experimental points from representative experiments. Figure 13 illustrates BMDC constitutively present exogenous soluble antigen on MHC class I molecules. BMDC or bone marrow-derived macrophages (5 x 10 5 ) were exposed to OVA in the presence (■; ♦) or absence (■; O) of 30 ng/ml PMA. The cells were washed and recultured for 2 hours before overlaying with 5 x 10 s B3ZT hybridoma cells. After 16 hours the cultures were either lysed and β-Gal activity measured using the CPRG substrate (absorbance max 595 nm) or fixed, incubated with X-gal, and the number of blue cells scored. Additional variables were either the OVA concentration (a) , BMDC (■; D) versus bone marrow-derived macrophages (♦, 0) (b) i time of exposure to antigen (c) , or chase period before fixation (d) . Background absorbance values obtained in the absence of OVA (ranging from 0.05 to 0.2 Au, or 0.2% Lac Z + cells, were substracted) . Readings shown are means of at least three independent experimental points. Figure 14 illustrates presentation of exogenous antigen utilizes the cytosolic processing machinery and early secretory pathway. (a) BMDC were incubated with 5 mg/ml OVA in the presence (■) or absence (D) of 1 mM gelonin for 10 min, then washed and incubated for 6 hours before overlaying with B3Z cells. (b & c) BMDC were incubated with 5 mg/ml OVA in the presence or absence of 5 μg/ml BFA (b) , 50 mM N-acetyl-L-Leu-L-L- norleucinal or N-acetyl-L-Leu-L-methinol (c) , as shown. BFA, N-acetyl-L-Leu-L-norleucinal, or N-acetyl-L-Leu-L- methional were added immediately after the antigen pulse and were present during the chase period up to 6 hours. In (b) , DC were fixed where indicated with 1% paraformaldehyde prior to co-culture with B3Z cells. Readings shown are the mean of three independent experimental points, with SEM shown as error bars. Figure 15 illustrates presentation of exogenous antigen is TAP-dependent in BMDC. DC expanded from B6 TAP +/+ mice (D) or TAP1 ' ' * mice (■) were incubated with either 5 mg/ml OVA for 10 min (a) or 1 mg/ml HRP for the times indicated (b) . Pinocytosis and antigen presentation were measured as described in Section 2.5 and 2.6. Cells exposed to HRP at 0°C was considered background loading. Triggering of B3Z T cells by TAPl"' " DC never increased above the background in the absence of OVA which is not subtracted in this experiment. Readings shown are means of at least three independent experimental points ± SEM. Figure 16 illustrates BMDC pulsed with OVA in vitro can prime CTL responses in vivo . BMDC incubated for 1 hour in the presence of 5 mg/ml OVA (D) , 10 nM SIINFEKL peptide (0) , or neither (Δ) , and 30 ng/ml PMA, and then washed. Under these conditions, presentation to B3Z was still completely TAP-dependent in vitro . After a 2 hour chase, 5 x 10 s cells were used to immunize B6 mice intravenously. After 9 days, spleens were removed and 2.5 x 10 7 responder cells restimulated with 1.2 x 10 7 syngeneic stimulator cells and 0.5 mM SIINFEKL peptide for 5 days. Cytotoxicity was measured in a conventional 4-h chromium release assay with RMA-S cells in the presence (a) or absence (b) of SIINFEKL peptide. Equivalent activity could be measured using EL4 and OVA-transfected EL4 (EG7.1) as targets. Bone marrow derived dendritic cells can capture and present exogenous antigens to T lymphocytes on class I MHC molecules following macropinocytosis of antigen, transfer of antigen to the cytosol and processing via the conventional class I pathway. These are key cells in the triggering of anti-viral and anti-tumour immune responses. The demonstration that they can trigger so called CD8 T cells following uptake of antigens from outside is important and exploitable. Key features of the pathway are: i) Using horseradish peroxidase (HRP) as a marker we show that approximately 30% of cells show cytosolic localisation of HRP after 15 minutes of exposure (Figure 11, a and b) . ii) Dendritic cells are considerably more efficient than macrophages isolated from the same bone marrow source. Importantly, although stimulation of macropinocytosis boosts antigen presentation and T cell activation, in dendritic cells this process occurs constitutively and this is sufficient for detectable levels of presentation (Fig 13) . iii) Presentation of exogenous antigen by dendritic cells is proteasome and TAP-dependent (Figs 14 and 15) which is the strongest evidence that antigen must gain access to the cytosol. iv) Dendritic cells exposed to antigen in vitro under conditions where macropinocytosis is maximally stimulated can upon injection into mice prime a T cell response in vivo (Fig 16) . This is an important step in demonstrating the physiological relevance of the pathway we have discovered and its possible therapeutic use. v) Recent experiments show that the membrane ruffling and macropinocytosis in dendritic cells can be blocked by microinjection of a so called dominant negative mutant of the GTP binding protein Rac (V12Nl&Rac) . This means that the ruffling and pinocytic response which leads to re-localisation of protein to the cytosol is controlled, as in other cells, by Rac and is therefore likely to be boosted further by activators of Rac. In other words novel compounds which activate Rac are likely to stimulate the overall process of cytosolic delivery. vi) A reporter cell line has been made which expresses the firefly luciferase gene under the control of a GAL4/VP16 activatable transcription unit. In the absence of GAL4/VP16 minimal luciferase is made. Upon expression or microinjection of GAL4/VP16 within the cells protein transcription of the luciferase gene is boosted dramatically. This is a relatively commonplace assay but the innovation is to add the GAL/VP16 protein from outside the cells under conditions where macropinocytosis is stimulated rather than expressing it within the cells. Basically, this would allow rapid screening of compounds for those which boost the membrane ruffling/macropinocytosis/cytosol delivery response. In summary, BMDC expanded in GM-CSF are able to capture, process, and present an exogenous antigen on MHC class I. Expression of the OVA epitope we have examined is strictly TAP-dependent and is blocked by BFA and proteasome inhibitors. Cytosolic access is achieved via constitutive membrane ruffling and formation of large macropinosomes, some of which deliver their content to the cytosol. Only a proportion of cells achieves visible access of exogenous tracers to the cytosol (Fig.2) in vitro , and expression of exogenous antigen on MHC class I appears to be substochiometric. In fact, this might be a desirable feature in vivo , since presentation of pathogen-encoded antigens on a limited number of professional APC can occur, even when those APC are note themselves infected. In other words, a few APC displaying peptides derived from exogenous antigens are used disposably to ensure CTL priming. Potentially, this route could be manipulated therapeutically to allow the generation of responses in vivo to tumours or cells infected with intracellular pathogens, where the dominant peptide-specific responses are not known. Discussion The disclosed herein demonstrates that macropinocytosed soluble antigen can gain access to the cytosol for presentation on class I MHC molecules. Presentation was regulated by the level of macropinocytic activity, utilised the conventional class I pathway and required no special antigen formulation. A pathway for the presentation of exogenous material on class I MHC molecules was predicted (Bevan, 1987; Townsend and Bodmer, 1989) based on reports of CD8 T cell priming by soluble proteins (Wraith et al., 9187; Staerz et al., 1987) and 'cross-priming' of host CD8 T cells to donor cell determinants restricted by MHC molecules found only on recipient bone marrow derived cells (Fink et al., 1983; Gooding and Edwards, 1980) . Such a pathway may also be necessary to explain how tumour-derived soluble heat shock proteins elicit tumour-specific, macrophage-dependent immunity (Udono et al., 1994), and how tumour cells induce CD8 responses restricted by the MHC of the host bone marrow rather than that of the inducing tumour cells (Huang et al. , 1994) . These in vivo observations suggest that there are exceptions to the general embargo on class I presentation of exogenous antigens. Usually, this blockade is considered necessary because cells that passively acquire CTL (cytotoxic T cell) determinant, but are not themselves infected, would be killed by activated CTLs recognising the exogenous material in the context of class I MHC molecules. However, it may be desirable to override this embargo in order to prime CTLs under certain conditions. For example, professional APCs (antigen presentation cells) are required to initiate most CTL responses (Lafferty et al., 1983; Mueller et al., 1989; Schwarts. , 1989; Ohashi et 1., 1991; Chen et al., 1992; Townsend and Allison. , 1993) suggesting that a pathogen may evade an immune response by not infecting or replicating within processional APCs or within cell types that traffic to lymphoid areas (Kundig, 1995) . In this case, the professional APC would not be able to use the conventional class I MHC pathway, which requires infection of the APC and subsequent de novo synthesis of pathogen-encoded proteins. In order to initiate a CTL response against such a pathogen, the professional APC would be required to present exogenous material on its class I MHC molecules. What features should such a pathway for presentation of exogenous antigen on class I MHC molecules have? First, it should be mostly confined to professional APC so that uninfected cells do not become targets for activated effector CD8 cells. Second, in contrast to the constitutive clathrin-mediated endocytic pathway, it should be up-regulatable to boost presentation of exogenous antigens in defined situations. Third, the loading of class I MHC should be 'cell-autonomous' in that only class I MHC molecules on the cell taking up antigen should be loaded to avoid passive transfer of material to 'bystander' cells. Fourth, the conventional proteasome- and TAP-dependent class I MHC pathway should be involved to ensure that CTLs are induced to epitopes that will be recognised when endogenously synthesised material is processed and presented. In other words T cells elicited by exogenous material must be useful as effector cells. The macropinocytic pathway described here is most active in professional antigen presenting cells and can be up and down-regulated (Swanson, 1989; Sallusto & Lanzavecchia, 1995) . Importantly, we have shown that presentation of soluble antigen on class II MHC molecules was similarly modulated upwards or downwards depending on ruffling and macropinocytic activity. Although we have no direct evidence that presentation on class I MHC molecules occurs via this pathway in vivo , we suggest that it constitutes a likely route for CTL induction by exogenous antigen. Interestingly, some bacteria stimulate macropinocytosis (Francis et al. , 1993) , resulting in non-phagocytic sequestration within macropinosomes (Alpuche-Aranda et al., 1994) . This observation raises the possibility that organisms which themselves have no obvious means of entering the cytosol, but to which CD8 T cell responses can nonetheless be made, might transfer material through macropinosome membranes into the cytosol. Yersinia (Starnbach and Bevan, 1994) and Listeria monocyotgenes mutants which lack listeriolysin (Szalay et al, 1994) constitute two possible examples. However, it should be stressed that several studies have demonstrated distinct non-classical loading of class I MHC molecules which is usually (Schirmbeck et al., 1995; Harding & Song, 1995) though not always (Liu et al., 1995; de Bruinj et al., 1995) brefelding A insensitive. Strikingly, the antigen preparations used are mostly particulate suggesting that the nature of the antigen may affect the pathway used. Thus, intracellular pathogens confined to vacuoles may load class I molecules at these sites following processing by endosomal rather than proteasomal proteases (Pfeiffer et al., 1993; de Bruijn et al., 1995) . The key point is that a good match between epitopes inducing CTLs and those presented by other cell types needs to be made. Perhaps two distinct pathways for loading class I MHC molecules with extracellular antigens exist: one for pathogens confined to vacuoles and a second, described here which may be most important for pathogen-derived class I determinants which are loaded via the conventional class I pathway. The present invention presents a pathway which allows unconventional access to the conventional class I MHC pathway in a regulatable and cell-type specific manner. The mechanism described here may constitute an important arm of the normal CD8 T cell response and may be useful for vaccination proposes. Modifications and improvements may be incorporated without departing from the scope of the invention. References The following documents are incorporated herein by reference. Apluche-Aranda, CM., Racoosin, E.L., Swanson, J.A. & Miller, S.I. (1994). J Exp Med 179 , 601-8. Bachmann, M.F., Kundig, T.M. , Freer, G. , Li, Y.,Kang, C.Y., Bishop, D.H., Hengartner, H. , Zinkernagel, R.M. (1994) . Eur. J. Immunol. 24, 2128-2236 Bevan, M.J. (1976). J.Exp.Med. 143 , 1283-1288. Bevan, M.J. (1987). Nature 325, 192-194. Blum-Tirouvanziam, U., Beghdadi-Rais, C , Roggero, M.A. , Valmori, D. , Bertholet, S., Bron, C, Fasel, N. , Corradin, G. (1994). J. Immunol. 153, 4134-4141. Chen., L. , Ashe, S. , Brady, W.A. , Hellstrom, I., Hellstrom, K.E., Ledbetter, J.A. , McGowan, P., Linsley, P.S. (1992). Cell 71 , 1093-1102. de Bruijn, M.L.H., Jackson, M.R. , Paterson, P.A. (1995). Eur. J. Immunol. 25 , 1274-1285. Dodi, A.I., Brett, S., Nordeng, T. , Sidhu, S., Batchelor, R.J. , Lombardi, G. , Bakke, O. , Lechler, R.I. (1994) . Eur. J. Immunol. 24, 1632-1639. Fink, P.J., Weissman, I.L. & Bevan, M.J. (1983) . J. Exp. Med. 157, 141-154. Francis, C . , Ryan, T.A. , Jones, B.D., Smith, S.J. & Falkow, S. (1993). Nature 364, 639-42. Germain, R.M. and Marguilies, D.H. (1993) . Annu. Rev. Immunol. 11, 403-500. Gooding, L.R. & Edwards, CB. (1980). J. Immunol. 124, 1258-1262. Harding, C.V. & Song, R. (1994). J. immunol 153, 4925- 33. Hewlett, L.J. (1995). Ph.D thesis. University of Dundee. Hewlett, L.J. , Prescott, A.R. & Watts, C (1994). J. Cell Biol. 124, 689-703. Huang, A.Y.C, Golumbek, P, Ahmadzadeh, M. , Jaffee, E. , Pardoll, D. & Levitsky, H. (1994). Science 264, 961- 965. Jaraquemada, D., Marti, M. , Long, E.O. (1990). J. Exp. Med, 172, 947-954. Karttunen, J., Sanderson, S. & Shastri, N. (1992) . Proc Natl Acad Sci USA 89, 6020-4. Ke, Y., Li, Y. , Kapp, J.A. (1995). Eur.J. Immunol. 25, 549-553. Kovacsovics-Bankowski, M. , Clark, K. , Benacerraf, B. & Rock, K.L. (1993). Proc Natl Acad Sci USA 90, 4942- 6. Kovacsovics-Bankowski, M. & rock, K.L. (1995) . Science 267, 243-6. Kundig, T.M ., Bach ann, M.F., DiPaolo, C, Simard, J.J.L., Battegay, M. , Lother, H. , Gessner, A., Kuhlcke, K. , Ohashi, P.S., Hengartner, H. , Zinkernagel, R. . (1995) . Science 268, 1343-1347. Lafferty, K.J. , Prowse, S.J. & Simeonovic CJ. (1983). Ann. Rev. Immunol 1, 143-173. Liu, T., Zhou, X., Orvell, C, Lederer, E. , Ljunggren, H-G., Jondal, M. (1995) J. Immunol. 154, 3147-3155. Moore, M.W. , Carbone, F.R. & Bevan, M.J. (1988) . Cell 54, 777-85. Morrison, L.A. , Lukacher A.E., Braciale, V.L, Fan, D.P. & Braciale, T.J. (1986) . J. Exp. Med. 163, 903-921. Nuchtern, J.G., Bonifacino, J.S., Biddison, W.E. & Klausner, R.D. (1989). Nature 339, 223-6. Ohashi, P.S., Oehen, S,m Buerki, K. , Pircher, H. , Ohashi, C, Odermatt, B., Malissen, B. , Zinkernagel, R. & Hengartner, H. (1991) . Cell 65, 305-317. Pfeifer, J.D., Wick, M.J., Roberts, R.L., Findlay, K. , Nor ark, S.J., Harding, C.V. (1993) . Nature 361, 359- 62. Racoosin, E.L. & Swanson, J.A. (1993). J.Cell Biol. 121, 1011-1020. Racoosin, E.L. & Swanson, J.A. (1989). J.Exp.Med. 170, 1635-48. Ridley, A.J., Paterson, H.F., Johnston, C.L., Diekmann, D., Hall, A. (1992) . Cell 70, 401-410. Rock, K.L., Gamble, S. , Rothstein, L. (1990). Science 249, 918-921. Rock, K.L., Gramm, C , Rothstein, L. , Clark, K. , Stein, R. , Dick, L. , Hwang, D. & Goldberg, A.L. (1994). Cell 78, 761-771. Rock, K.L., Rothstein, L. , Gamble, S. & Fleischacker, C (1993). C (1993). J. Immunol., 150, 438-446. Sallusto, F. , Cella, M. , Lanzavecchia, A. (1995). J.Exp.Med. in press. Sanderson, S. & Shastri, N. (1994). Int. Immunol. 6, 369-376. Sandvig, K. and van Deurs, B. (1994) Trends cell biol. 4, 275-277. Sasaki, T., Kishi, M. , Saito, M. , Tanaka, T. , Higuchi, N., Kominam, E., Katunuma, N. and Murachi, T. (1990). J. Enzyme Inhib. 3, 195-201. Schir beck, R. , Melber, K. , Rei ann, J. (1995) Eur. J. immunol. 25, 1063-1070. Schwartz, R.H. (1989) . Cell 57, 1073-1081. Staerz, U.D., Karasuyama, H. & Garner, A.M (1987) . Nature 329, 449-451. Starnbach, M.N. & Bevan, M.J. (1994) . J.Immunol. 153, 1603-1612. Stirpe, F., Olsnes, S. & Pihl, A. (1980). J. Biol. Chem.255, 6947-6953. Swanson, J.A., Yirinec, B.D. & Silverstein, S.C (1985). J. Cell Biol. 100, 851-859. Swanson, J.A. (1989). J Cell Sci 94, 135-42. Sugita, M. & Brenner, M.B. J. Biol. Chem. 270, 1443- 1448. Szalay, G., Hess, J. & Kaufmann, S.E. (1994). Eur.J.Immunol. 24, 1471-1477. Townsend, A.R.M. & Bodmer, H. (1989) . Ann. Rev. Immunol. 7, 601-624. Townsend, S.E. and Allison, J.P. (1993). Science 259, 368-370. Udono, H., Levey, D.L. & Srivastava, P.K. (1994). Proc.Natl. Acad.Sci. 91, 3077-3081. van Deurs, B., Sandvig, K., Peterson, O.W., olsnes, S. (1990) Int. Rev. Cytol. 117, 131-177. Watts, C. and Marsh, M. (1992). J.Cell Sci. 103, 1-8. West, M.A. , Bretscher, M.S. & Watts, C (1989). J Cell Biol 109, 2731-9. Wraith, D.C, Vessey, A.E. & Askonas, B.A. (1987) J.Gen.Virol. 68, 433-440. Yewdell, J. & Bennink, J.R. (1989) . Science 244, 1072- 1075. Zhou, X., Glas, R. , Liu, T. , Ljunggren, H-G. & Jondal, M. (1993). Eur. J. Immunol.23, 1802-1808.

Claims: 1 Claims 2 3 1. A method for the introduction of a substance into 4 living cells, the method comprising providing the 5 substance at a location adjacent to the cell 6 membrane, and allowing ingestion of the substance 7 into the cell by macropinocytosis. 8 9 2. A method as claimed in claim 1 comprising a step !0 of ruffling of the cell membrane. 11 12 3. A method as claimed in claims 1 or 2 wherein 13 agents which stimulate macropinocytosis and/or 14 ruffling are used. 15 16 4. A method as claimed in claim 3 comprising at least I *7 one stimulant chosen from the group comprising 18 phorbol esters such as PMA or MCSF (macrophage 19 colony stimulating factor) , growth factors such as 20 hepatocyte growth factor/scatter factor or 21 epidermal growth factor, bacterial or yeast cell 2 wall components agents for activation of the small 3 GTP binding protein rac, and other GTP binding 4 proteins. 5 6 5. A method as claimed in any of the preceding claims 7 wherein the substance to be introduced is linked 8 with a molecular signal to direct the matter to a 9 specific cellular target. 0 1 6. A method as claimed in claim 5 wherein the 2 molecular signal is a signal peptide. 3 4 7. A method as claimed in any of the preceding claims 5 wherein the substance to be introduced is a factor 6 which can modulate gene expression and the factor is directed to the nucleus by the signal peptide- CGGGPKKKRKVED. 8. A method as claimed in any of the preceding claims wherein the substance is chosen from a group of macromolecules including proteins, sugars, peptides, nucleic acids (RNA or DNA) , gene transcription factors, or any combination or complex of such macromolecules such as nucleic acid/protein complexes. 9. A method as claimed in any of the preceding claims wherein the substance is a DNA vaccine. 10. A method as claimed in any of the preceding claims wherein the substance is an agent for gene therapy. 11. Use of a method as claimed in claim 1 for the introduction of a substance and non-specific introduction of the substance into a cell. 12. Use of a method as claimed in any of claims 1 to ιo wherein the substance is introduced into the cytosol. 13. Use of a method as claimed in any of claims 1 to 10 to enhance the uptake of existing delivery vehicles such as liposomes and polymers. 14. Use of a method as claimed in any of claims 1 to 10 to introduce conventional or tumour antigens into antigen presenting cells to trigger lymphocytes which have anti-viral and anti-tumour characteristics when stimulated.

Inventors: WATTS COLIN, WATTS, COLIN
Applicants: UNIV DUNDEE, WATTS COLIN, THE UNIVERSITY COURT OF THE UNIVERSITY OF DUNDEE, WATTS, COLIN
Application year: 1996
Main classification: C12N15
IPC codes: A61K39, A61K47, A61K48