Vaccine 32 (2014) 2591–2598
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Heat treatment improves antigen-specific T cell activation after protein delivery by several but not all yeast genera Silvia Boschi Bazan a,1 , Tanja Breinig b,2 , Manfred J. Schmitt a , Frank Breinig a,∗ a b
Molecular and Cell Biology, Saarland University, 66123 Saarbrücken, Germany Junior Research Group for Virology/Immunology, Department of Virology, Saarland University Hospital, 66421 Homburg, Germany
a r t i c l e
i n f o
Article history: Received 10 July 2013 Received in revised form 10 January 2014 Accepted 13 March 2014 Available online 24 March 2014 Keywords: Yeast Vaccine T cell activation
a b s t r a c t A central prerequisite in using yeast as antigen carrier in vaccination is its efficient interaction with cellular components of the innate immune system, mainly mediated by cell surface structures. Here, we investigated the distribution of major yeast cell wall components such as mannan, -glucan and chitin of four different and likewise biotechnologically relevant yeasts (Saccharomyces, Pichia, Kluyveromyces and Schizosaccharomyces) and analyzed the influence of heat-treatment on -1,3-glucan exposure at the outer yeast cell surface as well as the amount of yeast induced reactive oxygen species (ROS) production by antigen presenting cells (APC) in human blood. We found that yeasts significantly differ in the distribution of their cell wall components and that heat-treatment affected both, cell wall composition and yeast-induced ROS production by human APCs. We further show that heat-treatment modulates the activation of antigen specific memory T cells after yeast-mediated protein delivery in different ways and thus provide additional support of using yeast as vehicle for the development of novel T cell vaccines. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Immune recognition of fungi mostly depends on the interaction of pattern recognition receptors (PRRs) on the surface of phagocytic cells with structural components on the fungal cell wall (pathogen-associated molecular patterns, PAMPs; reviewed by [1,2]). Generally, yeast cell walls consist of polysaccharides crosslinked to glycoproteins, and their major carbohydrate components are mannoproteins, -glucans and chitin [3,4]. The cell wall composition in fungi is not only species- and morphologydependent, it also represents a highly dynamic structure which is considerably remodeled during the yeast life cycle [1,3]. Several PRRs have been shown to associate with fungal PAMPs in a process dependent on cell type, fungal species and morphology [2]. All three major yeast cell wall components have been demonstrated to activate the mammalian immune system by acting as natural adjuvants [5–7]. Consequently, yeast has been used as
∗ Corresponding author at: Molecular and Cell Biology, Building A1.5, Saarland University, PO Box 151150, D-66123 Saarbrücken, Germany. Tel.: +49 681 302 2211; fax: +49 681 302 4710. E-mail address: [email protected] (F. Breinig). 1 Present address: Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, 05508-900 São Paulo, Brazil. 2 Present address: Computer Science, Saarland University, 66123 Saarbrücken, Germany. http://dx.doi.org/10.1016/j.vaccine.2014.03.043 0264-410X/© 2014 Elsevier Ltd. All rights reserved.
delivery vehicle for vaccines, aiming at inducing robust immune responses using either protein antigens or functional nucleic acids [8–11]. Fungal uptake by dendritic cells (DCs) has been described to promote DC maturation which in turn induces differentiation of naïve T cells into various subtypes of effector T helper or cytotoxic T cells (reviewed by [2]). We previously demonstrated that yeast-mediated antigen delivery is influenced by subcellular antigen localization and by the yeast vector itself [12]. Here we show that accessibility of yeast cell wall components affects antigen-specific T cell activation. We analyzed distribution patterns of major fungal cell wall constituents in different biotechnologically relevant yeasts before and after heat treatment, especially because heat-treated yeasts are used in most preclinical and clinical vaccination studies [13,14]. Furthermore, we show that heat treatment of intact fungal cells expressing the clinically relevant HCMV antigen pp65 influences the activation of memory T lymphocytes in different ways.
2. Materials and methods 2.1. Strains and media Escherichia coli DH5␣ was cultivated at 37 ◦ C in Luria-Bertani medium containing 100 g/mL ampicillin when appropriate. S. cerevisiae S86c (MAT˛ ura3-2 leu2 his3 pra1prb2 prc1 cps1; [15]), Sz.
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pombe PW260 (h-leu1.32 ura4. dl18 ade 6.210), K. lactis GG799 (wildtype isolate; New England Biolabs) and P. pastoris GS115 (his4; Invitrogen) were grown at 30 ◦ C in SC medium [12]. For recombinant protein production (30 ◦ C, 72 h), S. cerevisiae was cultured in ura d/o medium, Sz. pombe was cultivated in EMM [16] lacking leucine; P. pastoris was grown in BMG medium and shifted to BMM medium. 2.2. Immunolabelling of yeasts 100 l of an exponentially growing yeast culture were pelleted and washed with PBS (pH 7.4). For analysis of -1,3-glucan, cells were incubated 1 h with mouse anti--glucan (1:100; Biosupplies) specifically recognizing -1,3-glucans [17]. After washing with PBS, incubation with FITC-conjugated goat anti-mouse IgG (1:100; Sigma) was performed for 1 h in the dark. For detection of mannose or chitin, staining was performed with 200 g/ml of concanavalin A (ConA)-FITC (Invitrogen) or wheat germ agglutinin (WGA)-FITC (Sigma) [18], specifically binding ␣-mannan [19] or chitin oligomers on yeast cell walls [18,20,21]. Cells were washed with PBS and examined by both fluorescence microscopy (Keyence) and flow cytometry (FACS Calibur, BD Biosciences); data for 50,000 events were collected. Cut-off was adjusted to at least 97% FITCnegative control cells. 2.3. Measurement of ROS production in whole blood Exponentially growing yeast cells were harvested, washed with Hanks balanced salt solution (HBSS) and incubated at 65 ◦ C (heat treatment) for 1 h or left at room temperature. Heparinized human blood, diluted 1:10 in HBSS/0.1% gelatine, was incubated with 400 M luminol (diluted in DMSO) and 2.5 × 105 yeast cells (stimulated samples) or HBSS (resting samples). In blank control samples, blood was incubated with 2.5 × 105 yeast cells and DMSO. All samples were performed in triplicate in a Lumitrac plate (Greiner). Luminescence was recorded at 37 ◦ C using a plate reader (PARADIGM, Beckman Coulter). Data are expressed as relative luminescence units (RLU). Experiments were repeated three times. 2.4. Plasmids and yeast transformation Pp65 was PCR-amplified from plasmid pJW4303 with primers 5 pp65 (5 -CTCGAGATGATATCCGTACTGGGTCCCATTTC-3 ) and 3 pp65 (5 -AGATCTTCAACCTCGGTGCTTTTTGGGC-3 ), subcloned into pSTBlue-1 (Novagen) and cloned as EcoRI/BamHI fragment into S. cerevisiae expression vector pPGK [22]. S. cerevisiae S86c was transformed with pPGK/pp65 by the lithium acetate method [23] and transformants selected by uracil prototrophy. For cloning into the Sz. pombe expression vector pREP1 [24], pp65 was digested from pSTBlue-1 with XhoI/BamHI for inserting into SalI/BamHI-digested pREP1. Sz. pombe cells were transformed with pREP1/pp65 by a modified lithium acetate method [25] and selected on EMM agar plates lacking leucine containing 15 M thiamine. Cloning into the P. pastoris expression vector pPIC3.5 (Invitrogen) was performed by digesting pp65 with EcoRI/XbaI and ligating into EcoRI/AvrII restricted pPIC3.5. Electrocompetent P. pastoris GS115 cells (prepared according to supplier’s instructions [Invitrogen]) were electroporated with SalI-linearized pPIC3.5/pp65 and plated onto SC medium lacking histidine. 2.5. Western blot analysis 3 × 108 yeast cells were pelleted, washed with PBS and lysed with glass beads (0.5 mm). Pelleted cell debris and supernatants were precipitated with 2% sodium deoxycholate and trichloroacetic acid. Samples were electrophoresed on 10% SDS–polyacrylamide
gels, electrotransferred onto PVDF membranes (Roche) and incubated with mouse anti-pp65 (1:1000; Leica). Blots were treated with peroxidase-conjugated goat anti-mouse IgG (1:10,000; Sigma). Immunodetection was performed using the ECL system (Thermo Scientific). Recombinant pp65 was quantified by comparing the relative intensity of bands from 3 × 108 cells to a standard curve of commercially available pp65 (Miltenyi Biotec) of known concentration. Images were analyzed with QuantityOne software (Bio-Rad). 2.6. Whole blood assay Whole blood assays were performed as described with donor informed consent [26]. Heparinized human blood (450 l) was mixed with 1 g/ml of the costimulatory antibodies anti-CD28 and anti-CD49d (BD Biosciences). As positive control, 2.5 g/ml of Staphylococcus enterotoxin B (SEB; Sigma) were used; cells incubated with costimulatory antibodies only served as negative control. A lysate of HCMV-infected fibroblasts (Virion Serion) was used as positive control. Yeasts expressing recombinant pp65 or carrying control plasmids were washed with PBS and either heat-treated (65 ◦ C, 1 h) or left untreated. Stimulations were performed using 7.5 × 105 yeast cells/450 l blood. Samples were incubated at 37 ◦ C and 5% CO2 for 6 h, the last 4 h in the presence of Brefeldin A (10 g/ml). Cells were permeabilized with FACS buffer containing 0.1% saponin, immunostained using anti-CD4-PerCP (BD Biosciences), anti-CD8-PerCP (BD Biosciences), anti-IFN-␥-FITC (BD Biosciences) and anti-CD69-PE (Beckman Coulter), and analyzed using a FACScan (BD Biosciences). T cell stimulations were performed with blood from four HCMV-positive and three HCMVnegative donors. 3. Results 3.1. Distribution of major cell wall components among different yeast genera Previously, we demonstrated that several yeast genera currently investigated as antigen carriers are effectively endocytosed by human DCs [12]. However, since uptake of antigen carriers by APCs is triggered by engagement of PRRs on the APC to respective ligands on the carrier surface, we examined the accessibility of major fungal cell wall components in four biotechnologically relevant yeast species (S. cerevisiae, Sz. pombe, K. lactis and P. pastoris). Fluorescence microscopy showed varying mannan, -1,3-glucan and chitin staining patterns among the tested yeasts (Fig. 1A). Staining for yeast mannan revealed a relative homogeneous distribution over the cell surface in all yeasts (Fig. 1A, left panel), whereas 1,3-glucan localized to restricted regions of the cell walls of the budding yeasts S. cerevisiae, K. lactis and P. pastoris (Fig. 1A, central panel). In agreement with earlier findings [27], linear -1,3-glucan was exclusively restricted to the septum in fission yeast. Interestingly, chitin staining revealed a higher discrepancy among the yeast genera analyzed: P. pastoris clearly exhibited fluorescence around the cells, K. lactis and S. cerevisiae only in discrete patches. In contrast, Sz. pombe showed virtually no chitin fluorescence (Fig. 1A, right panel). Additional flow cytometric analyses confirmed that all yeast genera stained positive for mannan and -1,3-glucan, but not for chitin (Fig. 1B). The same pattern was observed for -1,3-glucan and chitin distribution: P. pastoris showed the highest median fluorescence intensity (MFI), followed by K. lactis, S. cerevisiae and Sz. pombe. Mannan MFI values were also higher in P. pastoris, followed by Sz. pombe, K. lactis and S. cerevisiae. Taken together, these results show that the tested yeast genera differ in accessibility of main cell wall components.
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Fig. 1. Distribution pattern of main cell wall components in different yeast genera. (A) Fluorescence microscopy after mannan, -1,3-glucan and chitin staining of S. cerevisiae, K. lactis, P. pastoris and Sz. pombe. Yeasts exponentially grown in SC medium were stained for mannan with Con A-FITC, for -1,3-glucan with a mouse anti--1,3-glucan antibody and anti-mouse IgG FITC as well as for chitin with WGA-FITC, and analyzed with a fluorescence microscope (GFP channel). Each column shows fluorescence (left) and matching bright field (right) micrographs; 50× zoom. (B) Flow cytometry of yeast cell wall components. Shown are the median fluorescence intensity (MFI) values from one representative experiment.
3.2. Influence of heat-treatment on ˇ-1,3-glucan distribution Observations that heat-killed yeasts expose more -1,3-glucan at the surface than untreated cells [28], together with the fact that heat-treated yeasts are used in some immunotherapy studies (reviewed by [13]), prompted us to investigate the effect of heating on yeast -1,3-glucan distribution. As shown in Fig. 2A, -1,3glucan localized in patch structures on the cell walls of untreated S. cerevisiae, K. lactis and P. pastoris. Thus, heat treatment increased fluorescence in budding yeasts, leading to a homogeneous staining of the entire cell wall. Nonetheless, in the case of untreated Sz. pombe cells, -1,3-glucan was exclusively found in the septum, which represents a typical phenomenon in this species [27]. However, after heating, fluorescence was not augmented but rather restricted to punctuate regions. Flow cytometric analyses (Fig. 2B) confirmed that heat-treated yeasts showed a clear increase in 1,3-glucan in S. cerevisiae, K. lactis and P. pastoris but not in Sz. pombe. Fig. 2B also shows that the highest increase in mean fluorescence of heat-treated cells was observed for S. cerevisiae, followed by K. lactis and P. pastoris. The opposite effect was observed for Sz. pombe in which -1,3-glucan levels were significantly reduced.
3.3. Yeast-induced reactive oxygen species (ROS) production in whole blood Since ROS production by phagocytes represents an important line in antifungal response [28], yeast cells were heated or left untreated and the production of ROS was determined in whole blood. All yeast genera were able to stimulate blood phagocytes, resulting in the production of ROS (Fig. 3). Chemiluminescence was detected approximately 6 min after addition of S. cerevisiae, K. lactis and P. pastoris cells, reaching a maximum after ∼30 min and declining thereafter; heat-treated cells increased chemiluminescence. Remarkably, untreated cells of fission yeast caused higher ROS production than heat-treated cells (Fig. 3). Moreover, chemiluminescence was detected after ∼20 min of incubation with Sz. pombe, reaching a maximum after 45 min for untreated yeasts, whereas incubation with heat-treated yeasts showed a different pattern, reaching a plateau after 64 min. This finding is in agreement with our previous studies, indicating a slower uptake of Sz. pombe by human phagocytes [12]. When either luminol or yeast were absent (blank or resting samples), no significant chemiluminescence was detected (Fig. 3). Thus, heat treatment exerts
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Fig. 2. Effect of heat-treatment on cell surface exposure of -1,3-glucan in different yeast genera. The indicated yeasts (S. cerevisiae, K. lactis, P. pastoris and Sz. pombe) were incubated at 65 ◦ C for 1 h or left untreated before being incubated with an anti--1,3-glucan antibody or an irrelevant antibody (isotype control). Then, cells were incubated with a FITC-coupled secondary antibody and analyzed by fluorescence microscopy or flow cytometry. (A) Fluorescence micrographs (left panel) and matching light micrographs (right panel) of cells stained for -1,3-glucan. (B) Histograms showing the influence of heat-treatment on -1,3-glucan exposure on the yeast cell surface. Dashed lines: cells incubated with an isotype control antibody (negative control); dotted lines: untreated cells; solid lines: heat-treated cells. The fold increase in fluorescence observed after heat treatment in comparison to non-treated cells is shown.
distinct effects on cell walls of different yeast species, leading to enhanced -1,3-glucan exposure and recognition on the cell surface in budding but not in fission yeast. 3.4. Activation of pp65-specific T lymphocytes in whole blood using recombinant yeast Next, we compared untreated or heat-treated whole yeasts with respect to their potential as protein delivery vector using pp65 of the human cytomegalovirus (HCMV) as model antigen. Expression of pp65 was analyzed by Western blotting. A single immunoreactive band was observed in yeast lysates of cells expressing pp65 and absent in extracts from yeasts carrying empty vectors. For unknown
reasons, pp65 expression was not detectable in K. lactis (data not shown). As shown in Fig. 4A, pp65 levels were highest in P. pastoris, followed by Sz. pombe and S. cerevisiae. Flow cytometric analyses of T cells from HCMV-positive donors revealed simultaneous IFN-␥/CD69 production in response to SEB (assay control) and to HCMV lysate (positive control). Incubation with costimulatory antibodies alone (negative control) did not cause significant effects on T cell activation (see Suppl. Fig. S1). In contrast, untreated or heat-treated pp65-expressing S. cerevisiae, Sz. pombe and P. pastoris activated significant amounts of antigen-specific memory CD4+ and CD8+ T lymphocytes (Fig. 4B). Heat-treated recombinant S. cerevisiae and P. pastoris were more efficient in activating both pp65-specific memory CD4+ and
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Fig. 3. ROS production detected by chemiluminescence kinetics of human whole blood depends on the extent of -1,3-glucan exposure on the yeast cell surface. Whole blood was incubated with heat-treated, untreated, or no yeast cells in the presence of luminol. Blank: blood samples incubated with yeasts and DMSO; resting: blood incubated with luminol without yeasts. Chemiluminescence was recorded at 37 ◦ C over a 150-min interval. RLU = relative luminescence units. Results represent mean values of triplicate determinations from one representative out of three experiments.
CD8+ T lymphocytes compared to untreated samples. In contrast, untreated Sz. pombe cells expressing pp65 caused a greater frequency of pp65-specific CD4+ and CD8+ T lymphocytes than heat-treated cells, whereas blood from HCMV seronegative donors did not stimulate pp65-specific lymphocytes (see Suppl. Fig. S1). In all donors, the responses against the yeast vehicles itselves were below 0.32/0.12% (CD4/CD8T cells, S. cerevisiae), 0.21/0.17% (P. pastoris), and 0.21/0.1% (Sz. pombe), respectively (see Suppl. Fig. S1). Taken together, our data indicate that heat treatment of S. cerevisiae and P. pastoris, but not Sz. pombe, leads to augmented antigenspecific CD4+ and CD8+ T cell activation. Notably, activation of antigen-specific memory T lymphocytes did not correlate with the amount of recombinant protein expressed by each yeast species, confirming our recent observation that the yeast vehicle itself can significantly influence cell-mediated immune responses [12]. Supplementary figure related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2014.03.043. 4. Discussion The recent use of whole yeasts as vectors for delivering protein antigens revealed a promising vaccine strategy, especially due to the natural adjuvant properties of yeast cell wall mannan, -glucan and chitin [13,14]. We focused on the distribution of these major cell wall components from yeast genera widely used in biotechnology by analyzing the influence of heating on -1,3-glucan fluctuations within the cell wall and the effect of increased -glucan exposure on both innate and adaptive immune responses. Our finding that the distribution of cell wall components significantly differs amongst budding (S. cerevisiae, P. pastoris and K. lactis) and fission yeast (Sz. pombe) is consistent with the marked structural dynamics and antigenic variability of the yeast cell wall (reviewed by [29]; [3,4]). Factors such as strain, age and type of growth medium have been described to influence yeast antigenicity, e.g. by altering the display of epitopes on the cell surface [29]. The composition of the mannan layer differs as a
consequence of diverse protein glycosylation patterns which in turn result from the divergence of the Golgi apparatus in the different yeast genera [30]. With respect to -1,3-glucan, budding has been shown to unmask this structure due to the generation of birth and bud scars [28] explaining the pronounced glucan staining in budding yeasts compared to fission yeast, which displays linear -1,3-glucan exclusively in the primary septum [27]. Chitin has been shown to associate with bud necks at the end of the cell cycle and becomes a characteristic feature of bud scars [31]. The presence of chitin in Sz. pombe is controversially discussed. Little amounts of this polymer have been described by some authors, while others failed to detect this polymer in fission yeast [32,33]. These differing distribution patterns in cell wall components and structures amongst yeasts may exert influence on how each yeast species is recognized by the mammalian immune system. According to previous studies, heat-treated cells of C. albicans and S. cerevisiae expose significantly more -glucan on the surface than untreated cells [28]. Upon heat treatment, matricial cell wall components are released, bringing -glucan to the outer layer and increasing the chance of phagocyte sensing through -glucan receptors [28,34]. In our study, heating of budding yeasts significantly enhanced the amount of exposed -1,3-glucan, while the same treatment had no effect in fission yeast. This finding was unexpected since linear -1,3-glucan in Sz. pombe is available in the primary septum [27]. -1,3-Glucan sensing through dectin-1 has been associated with stimulation of ROS production [35,36]. Hence, it was tempting to speculate that yeast cells subjected to heating would be more effective in eliciting ROS production due to augmented ligand availability. In fact, higher ROS production by bovine macrophages in response to heat-treated S. cerevisiae compared to untreated cells has been demonstrated [37]. In agreement with this, we found higher ROS production in human whole blood after incubation with heat-treated budding yeasts (S. cerevisiae, K. lactis and P. pastoris) compared to untreated cells. Noteworthy, the amount of ROS was lower when blood phagocytes were incubated with heat-treated Sz. pombe cells in comparison to untreated cells. Again, this observation was unanticipated, since the cell surface of this
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Fig. 4. Heterologous expression of HCMV pp65 in different yeasts and influence of heat-treatment on the activation of human pp65-specific memory T cells. (A) Westernblotting showing intracellular expression of pp65 by distinct yeast genera (left). Cell extracts corresponding to 3 × 108 yeast cells after 72 h of expression were precipitated with sodium deoxycholate/trichloroacetic acid and probed with a monoclonal anti-pp65 antibody. Yeast-derived 65 kDa protein bands identified as pp65 are shown. Lane 1: positive control (commercial pp65); lanes 2 and 3: lysates from S. cerevisiae cells harboring pPGK empty vector and pPGK/pp65, respectively; lanes 4 and 5: extracts from P. pastoris cells carrying pPIC3.5 and pPIC3.5/pp65, respectively; lanes 6 and 7: cell extracts from Sz. pombe carrying pREP1 vector and pREP1/pp65, respectively. Expression levels of recombinant pp65 in different yeast genera were determined (right). Quantification was performed based on a standard curve of purified recombinant pp65 of known concentration. Shown are mean values and standard deviations from triplicate determinations. (B) Yeasts expressing pp65 can activate antigen-specific memory T cells in whole blood of HCMV seropositive donors. Frequencies of antigen-specifically activated CD4 and CD8T cells after stimulation by pp65-expressing yeasts are shown. Antigens were added to whole blood of HCMV seropositive donors and stimulation was performed for 6 h. Activated T lymphocytes were identified by expression of CD69 and IFN-␥ using flow cytometry. To facilitate visualization of the antigen-specific response, results from samples incubated with yeasts carrying empty vectors were subtracted from those obtained with pp65-harboring yeasts. The corresponding primary data, including the controls, are shown in Suppl. Fig. S1.
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yeast contains -1,6-branched -1,3-glucan, which is also a target for dectin-1 [27,38]. These observations, together with our findings on -1,3-glucan staining intensity, indicate that heat treatment may alter the distribution of cell wall components in Sz. pombe in a different fashion than in budding yeasts. The next step was to investigate yeast-mediated delivery of a recombinant protein after heat treatment of whole fungal cells. Recombinant yeasts expressing the HCMV pp65 matrix protein were assessed for protein delivery efficiency in an ex vivo whole blood assay. Our group has previously shown that yeasts are rapidly internalized by blood phagocytes and efficiently processed for presentation to both CD4+ and CD8+ T lymphocytes [26,39]. Moreover, pp65 expressing fission yeast activated pp65-specific memory CD4+ and CD8+ T lymphocytes [26]. Here, we found that activation of antigen-specific memory T lymphocytes following yeast-mediated protein delivery increased after heat treatment of budding yeasts, but not of fission yeast. This observation cannot be attributed to varying pp65 expression levels in the different yeasts analyzed as Sz. pombe showed even higher expression levels than S. cerevisiae. Since heat treatment of budding yeasts, but not of fission yeast cells, leads to augmented exposure of the glucan component of the cell wall, this fact may suggest a link between the accessibility of -glucans and the activation of T cells. In fact, Ni et al. [40] showed the upregulation of T cell costimulatory molecules in human DCs followed by dectin-1 stimulation, potentiating antigen-specific CD8+ T cell responses [40]. Particulate yeast -glucan has been successfully used to stimulate robust cytotoxic T lymphocytes responses for combating tumors in animal models [41,42]. In addition, T cell responses were verified when particulate glucan from S. cerevisiae was used as vaccine delivery system [43,44], highlighting the role of -glucan in T cell activation. Interestingly, pp65-mediated delivery by untreated Sz. pombe cells elicited stronger activation of antigen-specific T lymphocytes than untreated P. pastoris and S. cerevisiae although the latter display considerably more -glucan on their cell surface; this may result from a most favorable combination of different cell wall constituents in fission yeast that positively influence its intrinsic adjuvant properties. A series of pre-clinical and clinical studies has resorted to using heat-inactivated yeast as protein delivery vector aiming at reducing potential risks involved in using live cells, particularly in immunocompromised individuals [13,14,45]. Our data suggest that genus and strain specific peculiarities in yeast cell wall composition should also be considered in the future design of yeast-based antigen delivery systems. Acknowledgements The authors thank Andreas Meyerhans for providing the plasmid pJW4303 containing a pp65 cDNA and Barbara Walch for technical advice. This work was supported by grants from the Alois Lauer Foundation (Dillingen, Germany) to TB and FB, and from CAPES (Brasília, Brazil) (BEX2767/07-4) to SBB and MJS. References [1] Brown GD. Innate antifungal immunity: the key role of phagocytes. Annu Rev Immunol 2011;29:1–21. [2] Romani L. Immunity to fungal infections. Nat Rev Immunol 2011;11:275–88. [3] Bowman SM, Free SJ. The structure and synthesis of the fungal cell wall. Bioessays 2006;28:799–808. [4] Levitz SM. Innate recognition of fungal cell walls. PLoS Pathog 2010;6:e1000758. [5] Da Silva CA, Pochard P, Lee CG, Elias JA. Chitin particles are multifaceted immune adjuvants. Am J Respir Crit Care Med 2010;182:1482–91. [6] Suzuki I, Hashimoto K, Ohno N, Tanaka H, Yadomae T. Immunomodulation by orally administered beta-glucan in mice. Int J Immunopharmacol 1989;11:761–9.
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Vaccine journal homepage: www.elsevier.com/locate/vaccine
Heat treatment improves antigen-specific T cell activation after protein delivery by several but not all yeast genera Silvia Boschi Bazan a,1 , Tanja Breinig b,2 , Manfred J. Schmitt a , Frank Breinig a,∗ a b
Molecular and Cell Biology, Saarland University, 66123 Saarbrücken, Germany Junior Research Group for Virology/Immunology, Department of Virology, Saarland University Hospital, 66421 Homburg, Germany
a r t i c l e
i n f o
Article history: Received 10 July 2013 Received in revised form 10 January 2014 Accepted 13 March 2014 Available online 24 March 2014 Keywords: Yeast Vaccine T cell activation
a b s t r a c t A central prerequisite in using yeast as antigen carrier in vaccination is its efficient interaction with cellular components of the innate immune system, mainly mediated by cell surface structures. Here, we investigated the distribution of major yeast cell wall components such as mannan, -glucan and chitin of four different and likewise biotechnologically relevant yeasts (Saccharomyces, Pichia, Kluyveromyces and Schizosaccharomyces) and analyzed the influence of heat-treatment on -1,3-glucan exposure at the outer yeast cell surface as well as the amount of yeast induced reactive oxygen species (ROS) production by antigen presenting cells (APC) in human blood. We found that yeasts significantly differ in the distribution of their cell wall components and that heat-treatment affected both, cell wall composition and yeast-induced ROS production by human APCs. We further show that heat-treatment modulates the activation of antigen specific memory T cells after yeast-mediated protein delivery in different ways and thus provide additional support of using yeast as vehicle for the development of novel T cell vaccines. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Immune recognition of fungi mostly depends on the interaction of pattern recognition receptors (PRRs) on the surface of phagocytic cells with structural components on the fungal cell wall (pathogen-associated molecular patterns, PAMPs; reviewed by [1,2]). Generally, yeast cell walls consist of polysaccharides crosslinked to glycoproteins, and their major carbohydrate components are mannoproteins, -glucans and chitin [3,4]. The cell wall composition in fungi is not only species- and morphologydependent, it also represents a highly dynamic structure which is considerably remodeled during the yeast life cycle [1,3]. Several PRRs have been shown to associate with fungal PAMPs in a process dependent on cell type, fungal species and morphology [2]. All three major yeast cell wall components have been demonstrated to activate the mammalian immune system by acting as natural adjuvants [5–7]. Consequently, yeast has been used as
∗ Corresponding author at: Molecular and Cell Biology, Building A1.5, Saarland University, PO Box 151150, D-66123 Saarbrücken, Germany. Tel.: +49 681 302 2211; fax: +49 681 302 4710. E-mail address: [email protected] (F. Breinig). 1 Present address: Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, 05508-900 São Paulo, Brazil. 2 Present address: Computer Science, Saarland University, 66123 Saarbrücken, Germany. http://dx.doi.org/10.1016/j.vaccine.2014.03.043 0264-410X/© 2014 Elsevier Ltd. All rights reserved.
delivery vehicle for vaccines, aiming at inducing robust immune responses using either protein antigens or functional nucleic acids [8–11]. Fungal uptake by dendritic cells (DCs) has been described to promote DC maturation which in turn induces differentiation of naïve T cells into various subtypes of effector T helper or cytotoxic T cells (reviewed by [2]). We previously demonstrated that yeast-mediated antigen delivery is influenced by subcellular antigen localization and by the yeast vector itself [12]. Here we show that accessibility of yeast cell wall components affects antigen-specific T cell activation. We analyzed distribution patterns of major fungal cell wall constituents in different biotechnologically relevant yeasts before and after heat treatment, especially because heat-treated yeasts are used in most preclinical and clinical vaccination studies [13,14]. Furthermore, we show that heat treatment of intact fungal cells expressing the clinically relevant HCMV antigen pp65 influences the activation of memory T lymphocytes in different ways.
2. Materials and methods 2.1. Strains and media Escherichia coli DH5␣ was cultivated at 37 ◦ C in Luria-Bertani medium containing 100 g/mL ampicillin when appropriate. S. cerevisiae S86c (MAT˛ ura3-2 leu2 his3 pra1prb2 prc1 cps1; [15]), Sz.
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pombe PW260 (h-leu1.32 ura4. dl18 ade 6.210), K. lactis GG799 (wildtype isolate; New England Biolabs) and P. pastoris GS115 (his4; Invitrogen) were grown at 30 ◦ C in SC medium [12]. For recombinant protein production (30 ◦ C, 72 h), S. cerevisiae was cultured in ura d/o medium, Sz. pombe was cultivated in EMM [16] lacking leucine; P. pastoris was grown in BMG medium and shifted to BMM medium. 2.2. Immunolabelling of yeasts 100 l of an exponentially growing yeast culture were pelleted and washed with PBS (pH 7.4). For analysis of -1,3-glucan, cells were incubated 1 h with mouse anti--glucan (1:100; Biosupplies) specifically recognizing -1,3-glucans [17]. After washing with PBS, incubation with FITC-conjugated goat anti-mouse IgG (1:100; Sigma) was performed for 1 h in the dark. For detection of mannose or chitin, staining was performed with 200 g/ml of concanavalin A (ConA)-FITC (Invitrogen) or wheat germ agglutinin (WGA)-FITC (Sigma) [18], specifically binding ␣-mannan [19] or chitin oligomers on yeast cell walls [18,20,21]. Cells were washed with PBS and examined by both fluorescence microscopy (Keyence) and flow cytometry (FACS Calibur, BD Biosciences); data for 50,000 events were collected. Cut-off was adjusted to at least 97% FITCnegative control cells. 2.3. Measurement of ROS production in whole blood Exponentially growing yeast cells were harvested, washed with Hanks balanced salt solution (HBSS) and incubated at 65 ◦ C (heat treatment) for 1 h or left at room temperature. Heparinized human blood, diluted 1:10 in HBSS/0.1% gelatine, was incubated with 400 M luminol (diluted in DMSO) and 2.5 × 105 yeast cells (stimulated samples) or HBSS (resting samples). In blank control samples, blood was incubated with 2.5 × 105 yeast cells and DMSO. All samples were performed in triplicate in a Lumitrac plate (Greiner). Luminescence was recorded at 37 ◦ C using a plate reader (PARADIGM, Beckman Coulter). Data are expressed as relative luminescence units (RLU). Experiments were repeated three times. 2.4. Plasmids and yeast transformation Pp65 was PCR-amplified from plasmid pJW4303 with primers 5 pp65 (5 -CTCGAGATGATATCCGTACTGGGTCCCATTTC-3 ) and 3 pp65 (5 -AGATCTTCAACCTCGGTGCTTTTTGGGC-3 ), subcloned into pSTBlue-1 (Novagen) and cloned as EcoRI/BamHI fragment into S. cerevisiae expression vector pPGK [22]. S. cerevisiae S86c was transformed with pPGK/pp65 by the lithium acetate method [23] and transformants selected by uracil prototrophy. For cloning into the Sz. pombe expression vector pREP1 [24], pp65 was digested from pSTBlue-1 with XhoI/BamHI for inserting into SalI/BamHI-digested pREP1. Sz. pombe cells were transformed with pREP1/pp65 by a modified lithium acetate method [25] and selected on EMM agar plates lacking leucine containing 15 M thiamine. Cloning into the P. pastoris expression vector pPIC3.5 (Invitrogen) was performed by digesting pp65 with EcoRI/XbaI and ligating into EcoRI/AvrII restricted pPIC3.5. Electrocompetent P. pastoris GS115 cells (prepared according to supplier’s instructions [Invitrogen]) were electroporated with SalI-linearized pPIC3.5/pp65 and plated onto SC medium lacking histidine. 2.5. Western blot analysis 3 × 108 yeast cells were pelleted, washed with PBS and lysed with glass beads (0.5 mm). Pelleted cell debris and supernatants were precipitated with 2% sodium deoxycholate and trichloroacetic acid. Samples were electrophoresed on 10% SDS–polyacrylamide
gels, electrotransferred onto PVDF membranes (Roche) and incubated with mouse anti-pp65 (1:1000; Leica). Blots were treated with peroxidase-conjugated goat anti-mouse IgG (1:10,000; Sigma). Immunodetection was performed using the ECL system (Thermo Scientific). Recombinant pp65 was quantified by comparing the relative intensity of bands from 3 × 108 cells to a standard curve of commercially available pp65 (Miltenyi Biotec) of known concentration. Images were analyzed with QuantityOne software (Bio-Rad). 2.6. Whole blood assay Whole blood assays were performed as described with donor informed consent [26]. Heparinized human blood (450 l) was mixed with 1 g/ml of the costimulatory antibodies anti-CD28 and anti-CD49d (BD Biosciences). As positive control, 2.5 g/ml of Staphylococcus enterotoxin B (SEB; Sigma) were used; cells incubated with costimulatory antibodies only served as negative control. A lysate of HCMV-infected fibroblasts (Virion Serion) was used as positive control. Yeasts expressing recombinant pp65 or carrying control plasmids were washed with PBS and either heat-treated (65 ◦ C, 1 h) or left untreated. Stimulations were performed using 7.5 × 105 yeast cells/450 l blood. Samples were incubated at 37 ◦ C and 5% CO2 for 6 h, the last 4 h in the presence of Brefeldin A (10 g/ml). Cells were permeabilized with FACS buffer containing 0.1% saponin, immunostained using anti-CD4-PerCP (BD Biosciences), anti-CD8-PerCP (BD Biosciences), anti-IFN-␥-FITC (BD Biosciences) and anti-CD69-PE (Beckman Coulter), and analyzed using a FACScan (BD Biosciences). T cell stimulations were performed with blood from four HCMV-positive and three HCMVnegative donors. 3. Results 3.1. Distribution of major cell wall components among different yeast genera Previously, we demonstrated that several yeast genera currently investigated as antigen carriers are effectively endocytosed by human DCs [12]. However, since uptake of antigen carriers by APCs is triggered by engagement of PRRs on the APC to respective ligands on the carrier surface, we examined the accessibility of major fungal cell wall components in four biotechnologically relevant yeast species (S. cerevisiae, Sz. pombe, K. lactis and P. pastoris). Fluorescence microscopy showed varying mannan, -1,3-glucan and chitin staining patterns among the tested yeasts (Fig. 1A). Staining for yeast mannan revealed a relative homogeneous distribution over the cell surface in all yeasts (Fig. 1A, left panel), whereas 1,3-glucan localized to restricted regions of the cell walls of the budding yeasts S. cerevisiae, K. lactis and P. pastoris (Fig. 1A, central panel). In agreement with earlier findings [27], linear -1,3-glucan was exclusively restricted to the septum in fission yeast. Interestingly, chitin staining revealed a higher discrepancy among the yeast genera analyzed: P. pastoris clearly exhibited fluorescence around the cells, K. lactis and S. cerevisiae only in discrete patches. In contrast, Sz. pombe showed virtually no chitin fluorescence (Fig. 1A, right panel). Additional flow cytometric analyses confirmed that all yeast genera stained positive for mannan and -1,3-glucan, but not for chitin (Fig. 1B). The same pattern was observed for -1,3-glucan and chitin distribution: P. pastoris showed the highest median fluorescence intensity (MFI), followed by K. lactis, S. cerevisiae and Sz. pombe. Mannan MFI values were also higher in P. pastoris, followed by Sz. pombe, K. lactis and S. cerevisiae. Taken together, these results show that the tested yeast genera differ in accessibility of main cell wall components.
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Fig. 1. Distribution pattern of main cell wall components in different yeast genera. (A) Fluorescence microscopy after mannan, -1,3-glucan and chitin staining of S. cerevisiae, K. lactis, P. pastoris and Sz. pombe. Yeasts exponentially grown in SC medium were stained for mannan with Con A-FITC, for -1,3-glucan with a mouse anti--1,3-glucan antibody and anti-mouse IgG FITC as well as for chitin with WGA-FITC, and analyzed with a fluorescence microscope (GFP channel). Each column shows fluorescence (left) and matching bright field (right) micrographs; 50× zoom. (B) Flow cytometry of yeast cell wall components. Shown are the median fluorescence intensity (MFI) values from one representative experiment.
3.2. Influence of heat-treatment on ˇ-1,3-glucan distribution Observations that heat-killed yeasts expose more -1,3-glucan at the surface than untreated cells [28], together with the fact that heat-treated yeasts are used in some immunotherapy studies (reviewed by [13]), prompted us to investigate the effect of heating on yeast -1,3-glucan distribution. As shown in Fig. 2A, -1,3glucan localized in patch structures on the cell walls of untreated S. cerevisiae, K. lactis and P. pastoris. Thus, heat treatment increased fluorescence in budding yeasts, leading to a homogeneous staining of the entire cell wall. Nonetheless, in the case of untreated Sz. pombe cells, -1,3-glucan was exclusively found in the septum, which represents a typical phenomenon in this species [27]. However, after heating, fluorescence was not augmented but rather restricted to punctuate regions. Flow cytometric analyses (Fig. 2B) confirmed that heat-treated yeasts showed a clear increase in 1,3-glucan in S. cerevisiae, K. lactis and P. pastoris but not in Sz. pombe. Fig. 2B also shows that the highest increase in mean fluorescence of heat-treated cells was observed for S. cerevisiae, followed by K. lactis and P. pastoris. The opposite effect was observed for Sz. pombe in which -1,3-glucan levels were significantly reduced.
3.3. Yeast-induced reactive oxygen species (ROS) production in whole blood Since ROS production by phagocytes represents an important line in antifungal response [28], yeast cells were heated or left untreated and the production of ROS was determined in whole blood. All yeast genera were able to stimulate blood phagocytes, resulting in the production of ROS (Fig. 3). Chemiluminescence was detected approximately 6 min after addition of S. cerevisiae, K. lactis and P. pastoris cells, reaching a maximum after ∼30 min and declining thereafter; heat-treated cells increased chemiluminescence. Remarkably, untreated cells of fission yeast caused higher ROS production than heat-treated cells (Fig. 3). Moreover, chemiluminescence was detected after ∼20 min of incubation with Sz. pombe, reaching a maximum after 45 min for untreated yeasts, whereas incubation with heat-treated yeasts showed a different pattern, reaching a plateau after 64 min. This finding is in agreement with our previous studies, indicating a slower uptake of Sz. pombe by human phagocytes [12]. When either luminol or yeast were absent (blank or resting samples), no significant chemiluminescence was detected (Fig. 3). Thus, heat treatment exerts
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Fig. 2. Effect of heat-treatment on cell surface exposure of -1,3-glucan in different yeast genera. The indicated yeasts (S. cerevisiae, K. lactis, P. pastoris and Sz. pombe) were incubated at 65 ◦ C for 1 h or left untreated before being incubated with an anti--1,3-glucan antibody or an irrelevant antibody (isotype control). Then, cells were incubated with a FITC-coupled secondary antibody and analyzed by fluorescence microscopy or flow cytometry. (A) Fluorescence micrographs (left panel) and matching light micrographs (right panel) of cells stained for -1,3-glucan. (B) Histograms showing the influence of heat-treatment on -1,3-glucan exposure on the yeast cell surface. Dashed lines: cells incubated with an isotype control antibody (negative control); dotted lines: untreated cells; solid lines: heat-treated cells. The fold increase in fluorescence observed after heat treatment in comparison to non-treated cells is shown.
distinct effects on cell walls of different yeast species, leading to enhanced -1,3-glucan exposure and recognition on the cell surface in budding but not in fission yeast. 3.4. Activation of pp65-specific T lymphocytes in whole blood using recombinant yeast Next, we compared untreated or heat-treated whole yeasts with respect to their potential as protein delivery vector using pp65 of the human cytomegalovirus (HCMV) as model antigen. Expression of pp65 was analyzed by Western blotting. A single immunoreactive band was observed in yeast lysates of cells expressing pp65 and absent in extracts from yeasts carrying empty vectors. For unknown
reasons, pp65 expression was not detectable in K. lactis (data not shown). As shown in Fig. 4A, pp65 levels were highest in P. pastoris, followed by Sz. pombe and S. cerevisiae. Flow cytometric analyses of T cells from HCMV-positive donors revealed simultaneous IFN-␥/CD69 production in response to SEB (assay control) and to HCMV lysate (positive control). Incubation with costimulatory antibodies alone (negative control) did not cause significant effects on T cell activation (see Suppl. Fig. S1). In contrast, untreated or heat-treated pp65-expressing S. cerevisiae, Sz. pombe and P. pastoris activated significant amounts of antigen-specific memory CD4+ and CD8+ T lymphocytes (Fig. 4B). Heat-treated recombinant S. cerevisiae and P. pastoris were more efficient in activating both pp65-specific memory CD4+ and
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Fig. 3. ROS production detected by chemiluminescence kinetics of human whole blood depends on the extent of -1,3-glucan exposure on the yeast cell surface. Whole blood was incubated with heat-treated, untreated, or no yeast cells in the presence of luminol. Blank: blood samples incubated with yeasts and DMSO; resting: blood incubated with luminol without yeasts. Chemiluminescence was recorded at 37 ◦ C over a 150-min interval. RLU = relative luminescence units. Results represent mean values of triplicate determinations from one representative out of three experiments.
CD8+ T lymphocytes compared to untreated samples. In contrast, untreated Sz. pombe cells expressing pp65 caused a greater frequency of pp65-specific CD4+ and CD8+ T lymphocytes than heat-treated cells, whereas blood from HCMV seronegative donors did not stimulate pp65-specific lymphocytes (see Suppl. Fig. S1). In all donors, the responses against the yeast vehicles itselves were below 0.32/0.12% (CD4/CD8T cells, S. cerevisiae), 0.21/0.17% (P. pastoris), and 0.21/0.1% (Sz. pombe), respectively (see Suppl. Fig. S1). Taken together, our data indicate that heat treatment of S. cerevisiae and P. pastoris, but not Sz. pombe, leads to augmented antigenspecific CD4+ and CD8+ T cell activation. Notably, activation of antigen-specific memory T lymphocytes did not correlate with the amount of recombinant protein expressed by each yeast species, confirming our recent observation that the yeast vehicle itself can significantly influence cell-mediated immune responses [12]. Supplementary figure related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2014.03.043. 4. Discussion The recent use of whole yeasts as vectors for delivering protein antigens revealed a promising vaccine strategy, especially due to the natural adjuvant properties of yeast cell wall mannan, -glucan and chitin [13,14]. We focused on the distribution of these major cell wall components from yeast genera widely used in biotechnology by analyzing the influence of heating on -1,3-glucan fluctuations within the cell wall and the effect of increased -glucan exposure on both innate and adaptive immune responses. Our finding that the distribution of cell wall components significantly differs amongst budding (S. cerevisiae, P. pastoris and K. lactis) and fission yeast (Sz. pombe) is consistent with the marked structural dynamics and antigenic variability of the yeast cell wall (reviewed by [29]; [3,4]). Factors such as strain, age and type of growth medium have been described to influence yeast antigenicity, e.g. by altering the display of epitopes on the cell surface [29]. The composition of the mannan layer differs as a
consequence of diverse protein glycosylation patterns which in turn result from the divergence of the Golgi apparatus in the different yeast genera [30]. With respect to -1,3-glucan, budding has been shown to unmask this structure due to the generation of birth and bud scars [28] explaining the pronounced glucan staining in budding yeasts compared to fission yeast, which displays linear -1,3-glucan exclusively in the primary septum [27]. Chitin has been shown to associate with bud necks at the end of the cell cycle and becomes a characteristic feature of bud scars [31]. The presence of chitin in Sz. pombe is controversially discussed. Little amounts of this polymer have been described by some authors, while others failed to detect this polymer in fission yeast [32,33]. These differing distribution patterns in cell wall components and structures amongst yeasts may exert influence on how each yeast species is recognized by the mammalian immune system. According to previous studies, heat-treated cells of C. albicans and S. cerevisiae expose significantly more -glucan on the surface than untreated cells [28]. Upon heat treatment, matricial cell wall components are released, bringing -glucan to the outer layer and increasing the chance of phagocyte sensing through -glucan receptors [28,34]. In our study, heating of budding yeasts significantly enhanced the amount of exposed -1,3-glucan, while the same treatment had no effect in fission yeast. This finding was unexpected since linear -1,3-glucan in Sz. pombe is available in the primary septum [27]. -1,3-Glucan sensing through dectin-1 has been associated with stimulation of ROS production [35,36]. Hence, it was tempting to speculate that yeast cells subjected to heating would be more effective in eliciting ROS production due to augmented ligand availability. In fact, higher ROS production by bovine macrophages in response to heat-treated S. cerevisiae compared to untreated cells has been demonstrated [37]. In agreement with this, we found higher ROS production in human whole blood after incubation with heat-treated budding yeasts (S. cerevisiae, K. lactis and P. pastoris) compared to untreated cells. Noteworthy, the amount of ROS was lower when blood phagocytes were incubated with heat-treated Sz. pombe cells in comparison to untreated cells. Again, this observation was unanticipated, since the cell surface of this
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Fig. 4. Heterologous expression of HCMV pp65 in different yeasts and influence of heat-treatment on the activation of human pp65-specific memory T cells. (A) Westernblotting showing intracellular expression of pp65 by distinct yeast genera (left). Cell extracts corresponding to 3 × 108 yeast cells after 72 h of expression were precipitated with sodium deoxycholate/trichloroacetic acid and probed with a monoclonal anti-pp65 antibody. Yeast-derived 65 kDa protein bands identified as pp65 are shown. Lane 1: positive control (commercial pp65); lanes 2 and 3: lysates from S. cerevisiae cells harboring pPGK empty vector and pPGK/pp65, respectively; lanes 4 and 5: extracts from P. pastoris cells carrying pPIC3.5 and pPIC3.5/pp65, respectively; lanes 6 and 7: cell extracts from Sz. pombe carrying pREP1 vector and pREP1/pp65, respectively. Expression levels of recombinant pp65 in different yeast genera were determined (right). Quantification was performed based on a standard curve of purified recombinant pp65 of known concentration. Shown are mean values and standard deviations from triplicate determinations. (B) Yeasts expressing pp65 can activate antigen-specific memory T cells in whole blood of HCMV seropositive donors. Frequencies of antigen-specifically activated CD4 and CD8T cells after stimulation by pp65-expressing yeasts are shown. Antigens were added to whole blood of HCMV seropositive donors and stimulation was performed for 6 h. Activated T lymphocytes were identified by expression of CD69 and IFN-␥ using flow cytometry. To facilitate visualization of the antigen-specific response, results from samples incubated with yeasts carrying empty vectors were subtracted from those obtained with pp65-harboring yeasts. The corresponding primary data, including the controls, are shown in Suppl. Fig. S1.
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yeast contains -1,6-branched -1,3-glucan, which is also a target for dectin-1 [27,38]. These observations, together with our findings on -1,3-glucan staining intensity, indicate that heat treatment may alter the distribution of cell wall components in Sz. pombe in a different fashion than in budding yeasts. The next step was to investigate yeast-mediated delivery of a recombinant protein after heat treatment of whole fungal cells. Recombinant yeasts expressing the HCMV pp65 matrix protein were assessed for protein delivery efficiency in an ex vivo whole blood assay. Our group has previously shown that yeasts are rapidly internalized by blood phagocytes and efficiently processed for presentation to both CD4+ and CD8+ T lymphocytes [26,39]. Moreover, pp65 expressing fission yeast activated pp65-specific memory CD4+ and CD8+ T lymphocytes [26]. Here, we found that activation of antigen-specific memory T lymphocytes following yeast-mediated protein delivery increased after heat treatment of budding yeasts, but not of fission yeast. This observation cannot be attributed to varying pp65 expression levels in the different yeasts analyzed as Sz. pombe showed even higher expression levels than S. cerevisiae. Since heat treatment of budding yeasts, but not of fission yeast cells, leads to augmented exposure of the glucan component of the cell wall, this fact may suggest a link between the accessibility of -glucans and the activation of T cells. In fact, Ni et al. [40] showed the upregulation of T cell costimulatory molecules in human DCs followed by dectin-1 stimulation, potentiating antigen-specific CD8+ T cell responses [40]. Particulate yeast -glucan has been successfully used to stimulate robust cytotoxic T lymphocytes responses for combating tumors in animal models [41,42]. In addition, T cell responses were verified when particulate glucan from S. cerevisiae was used as vaccine delivery system [43,44], highlighting the role of -glucan in T cell activation. Interestingly, pp65-mediated delivery by untreated Sz. pombe cells elicited stronger activation of antigen-specific T lymphocytes than untreated P. pastoris and S. cerevisiae although the latter display considerably more -glucan on their cell surface; this may result from a most favorable combination of different cell wall constituents in fission yeast that positively influence its intrinsic adjuvant properties. A series of pre-clinical and clinical studies has resorted to using heat-inactivated yeast as protein delivery vector aiming at reducing potential risks involved in using live cells, particularly in immunocompromised individuals [13,14,45]. Our data suggest that genus and strain specific peculiarities in yeast cell wall composition should also be considered in the future design of yeast-based antigen delivery systems. Acknowledgements The authors thank Andreas Meyerhans for providing the plasmid pJW4303 containing a pp65 cDNA and Barbara Walch for technical advice. This work was supported by grants from the Alois Lauer Foundation (Dillingen, Germany) to TB and FB, and from CAPES (Brasília, Brazil) (BEX2767/07-4) to SBB and MJS. References [1] Brown GD. Innate antifungal immunity: the key role of phagocytes. Annu Rev Immunol 2011;29:1–21. [2] Romani L. Immunity to fungal infections. Nat Rev Immunol 2011;11:275–88. [3] Bowman SM, Free SJ. The structure and synthesis of the fungal cell wall. Bioessays 2006;28:799–808. [4] Levitz SM. Innate recognition of fungal cell walls. PLoS Pathog 2010;6:e1000758. [5] Da Silva CA, Pochard P, Lee CG, Elias JA. Chitin particles are multifaceted immune adjuvants. Am J Respir Crit Care Med 2010;182:1482–91. [6] Suzuki I, Hashimoto K, Ohno N, Tanaka H, Yadomae T. Immunomodulation by orally administered beta-glucan in mice. Int J Immunopharmacol 1989;11:761–9.
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