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Extensive MHC class I-restricted CD8 T lymphocyte responses against various yeast genera in humans
FEMS Immunology and Medical Microbiology 39 (2003) 279^286
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Extensive MHC class I-restricted CD8 T lymphocyte responses against various yeast genera in humans Tanja Heintel a
a;1
, Frank Breinig
b;1
, Manfred J. Schmitt b , Andreas Meyerhans
a;
Department of Virology, Institute of Medical Microbiology and Hygiene, Building 47, University of the Saarland, 66421 Homburg, Germany b Department of Applied Molecular Biology, University of the Saarland, 66123 Saarbru«cken, Germany Received 9 May 2003; received in revised form 20 August 2003; accepted 13 September 2003 First published online 21 October 2003
Abstract The human cellular immune response against 14 distantly related yeast species was analyzed by intracellular cytokine staining of lymphocytes after ex vivo stimulation of whole blood. While the CD4 T cell response was marginal, extensive MHC class I-restricted CD8 T cell responses were detected against a number of species including spoiling, environmental and human pathogenic yeasts. The yeastspecific CD8 T cells expressed interferon-Q but lacked expression of CD27 and CCR7, indicating that they were end-differentiated effector memory cells. Mainly intact yeast cells rather than spheroplasts were able to induce cytokine expression in T cells demonstrating that the dominant immunogens were located in the yeast cell wall. Together these data underline the importance of the cellular immune response in protecting humans against yeast and fungal infections. And, from another perspective, recombinant yeast suggests itself as a potential vaccine candidate to efficiently induce antigen-specific CD8 T cell responses. 5 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords : Human ; Fungal infection ; Cellular activation; T lymphocyte
1. Introduction Yeast species are unicellular fungi, most of them classi¢ed within the ascomycetes. They usually £ourish in habitats like fruits, £owers and the bark of trees and thus exist in the close surroundings of humans. In particular Saccharomyces cerevisiae, which is known as baker’s or brewer’s yeast, has been used for centuries for the production of bread and alcoholic beverages. Likewise, Schizosaccharomyces pombe is used for brewing of millet beer in Africa. A number of yeast species live in symbiosis with animals and pathogenicity in humans is rare. However, individuals with an impaired immune system due to immunosuppressive drugs or infections caused by agents including human immunode¢ciency virus (HIV), may experience life-threatening systemic infections with pathogenic strains of Cryptococcus neoformans and Candida albicans [1^4].
* Corresponding author. Tel. : +49 (6841) 162 3990 ; Fax : +49 (6841) 162 3980. E-mail address : [email protected] (A. Meyerhans). 1
These authors contributed equally to this work.
Both innate and acquired immune responses participate in the protection against yeast infections. Polymorphonuclear neutrophils were shown to play a critical role in providing early resistance against the human pathogenic yeasts C. neoformans and Paracoccidioides brasiliensis [5,6]. In untreated HIV-infected patients with advanced immunode¢ciency, such leukocytes have diminished antimicrobial and secretory functions which result in a defect in protection against C. neoformans and other opportunistic pathogens [3,7,8]. For the two major human pathogenic yeasts, C. neoformans and C. albicans, protective as well as non-protective antibodies have been identi¢ed, suggesting that the e⁄cacy of antibody-mediated protection is a function of isotype, dose and speci¢city [9,10]. Adoptive lymphocyte transfer experiments in mice have shown that cellular immunity can also mediate protection against several yeast species while defects in cellular immune responses enhance the susceptibility towards such infections [11,12]. Regarding the T helper (Th) phenotype, Th1 cells usually result in resistance while Th2 cell responses are associated with susceptibility to C. albicans and C. neoformans infections [12^14]. Until today, most experiments on cellular immune responses against yeast refer to human pathogenic yeast
0928-8244 / 03 / $22.00 5 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0928-8244(03)00294-3
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species. To globally characterize the human T cell response against yeast, we have investigated the stimulation of T lymphocytes by a variety of distantly related yeast genera from di¡erent habitats, including spoiling, environmental, opportunistically pathogenic as well as traditionally cultured yeasts. An ex vivo whole blood assay was performed that permits the identi¢cation and simultaneous quanti¢cation of antigen-speci¢c CD4 and CD8 T cells as already described for HIV- and CMV-speci¢c T cells [15^ 17]. Whole blood is incubated for 6 h with the respective antigens. These are taken up by antigen presenting cells within the blood and subsequently enter the pathways of antigen processing and presentation [18]. Interestingly, the processed peptides are presented by major histocompatibility proteins of classes I and II, thus allowing to measure MHC class I-dependent CD8 T cell activation and MHC class II-dependent CD4 T cell activation. The speci¢c T cell stimulation then results in the upregulation of the early activation marker CD69 and the production of interferon-Q (IFN-Q) and other cytokines which can be quanti¢ed by £ow cytometry [16,18]. During the 6-h incubation time used here, only e¡ector and memory CD4 and CD8 T cells are speci¢cally stimulated, whereas the di¡erentiation kinetics of naive T cells do not allow such a rapid activation [19,20]. Therefore the whole blood assay under our conditions is able to quantify pre-existing cellular immune responses. Here we show that an e⁄cient uptake of intact yeast cells by phagocytosis is followed by an extensive yeastspeci¢c and MHC class I-restricted immune response of CD8 T lymphocytes. The immunogens inducing the CD8 T cell responses are located within the yeast cell wall. Moreover, these yeast-speci¢c CD8 T cells are characterized by an end-di¡erentiated e¡ector memory phenotype, because they show the typical lack of the di¡erentiation markers CD27 and CCR7 [21,22]. Together these data underline the potential of recombinant yeast in the development of novel vaccines inducing HLA class I-restricted CD8 T cell responses.
2. Materials and methods 2.1. Stimulation of yeast-speci¢c CD4 and CD8 T cells within whole blood Antigen stimulation of yeast-speci¢c T cells was performed in whole blood as described previously [16]. Blood donors were ¢ve healthy European individuals, three females and two males around 30 years of age. For some experiments blood from arbitrarily chosen donors among these was used. Brie£y, 450 Wl of heparinized blood were mixed with 1 Wg ml31 of the costimulatory antibodies KCD28 and KCD49d each (clones L293 and L25.3, BD Pharmingen, Heidelberg, Germany). Stimulations were carried out using titrated amounts of yeast cells
(7.5U105 cells per 450 Wl blood). To obtain optimal T cell responses, titration of the yeast cell amount was performed with 7.5U102 to 7.5U106 cells of the highly immunogenic yeast Williopsis californica (not shown). As negative control, cells were stimulated with costimulatory antibodies only. The whole blood was incubated in 15-ml polypropylene tubes at 37‡C/5% CO2 for a total of 6 h. During the last 4 h 10 Wg ml31 of Brefeldin A (Sigma, Deisenhofen, Germany) was added to inhibit the cytokine secretion. After incubation of the blood with 2 mM ethylenediamine tetraacetic acid (EDTA) for 15 min, erythrocytes were lysed and leukocytes were ¢xed for 10 min using Becton Dickinson lysing solution according to the manufacturer’s instruction. Thereafter, cells were washed once with FACS bu¡er (phosphate-bu¡ered saline (PBS), 5% ¢ltered fetal calf serum (FCS), 0.5% bovine serum albumin (BSA), 0.07% NaN3 ) and left overnight at 4‡C. For the determination of the frequency of antigen-speci¢c CD4 and CD8 T cells by £ow cytometry, the ¢xed leukocytes were permeabilized with 2 ml FACS bu¡er containing 0.1% saponin (Sigma) for 10 min at room temperature (RT). The immunostaining was carried out for 30 min at RT in the dark using saturating conditions of the antibodies anti-CD4 and anti-CD8 (clone SK3 and clone SK1, PerCP, Becton Dickinson, Heidelberg, Germany), anti-IFN-Q (clone 4S.B3, £uorescein isothiocyanate (FITC) or phenylethylamine (PE), BD Pharmingen) or anti-CD69 (clone TP1.55.3, PE, Beckman Coulter, Krefeld, Germany). For a further characterization of yeastspeci¢c T cells, the antibodies anti-CD27 (clone M-T271, FITC, BD Pharmingen), anti-CCR7 (clone 3D12, Max Delbru«ck Centrum) and donkey anti-rat (PE, Dianova, Hamburg, Germany) were used. Cells were washed once with FACS bu¡er and ¢xed using 1% paraformaldehyde. CD4- and CD8-positive T cells were identi¢ed by gating on the lymphocyte population via cell size and granularity and their high CD4 and CD8 expression level, respectively. At least 25 000 CD4- and CD8-positive lymphocytes were analyzed on a FACScan (Becton Dickinson) using the Cellquest Software. Speci¢cally activated T lymphocytes were identi¢ed and quanti¢ed as CD69 and IFN-Q double-positive cells. The addition of costimulatory antibodies only (negative control) led not to a signi¢cant T cell stimulation (frequencies below 0.05%). 2.2. Yeast-speci¢c stimulation of peripheral blood mononuclear cells (PBMC) Human PBMC were isolated from fresh, heparinized blood through density gradient centrifugation over Ficoll-Hypaque (Pharmacia, Freiburg, Germany). Antigen stimulations were carried out in polypropylene FACS tubes using 2U106 cells in 400 Wl RPMI+5% human serum (Sigma) per sample. Costimulatory antibodies and yeast cells were added as mentioned above. After an incubation period of 30 min at 37‡C/5% CO2 , the samples were cen-
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trifuged for 5 min at 1100 rpm and incubated for another 90 min. This centrifugation step was repeated after the subsequent addition of Brefeldin A. The cells were ¢rst incubated with 2 mM EDTA for 15 min (see above), washed with 2 ml PBS+0.02% EDTA and ¢xed with 4% paraformaldehyde for 5 min at RT. Fixation was stopped by the addition of FACS bu¡er. 2.3. Yeast genera, yeast culture and spheroplast preparation The various yeast genera used in this study were provided by the yeast strain collection of the Institute of Applied Molecular Biology, Saarland University. All yeast strains were grown in YEPD medium (2% glucose, 2% peptone, 1% yeast extract) at 30‡C. Before adding to the whole blood (see above), yeasts were washed three times in PBS (5000 rpm, 10 min, RT). For C. albicans, four di¡erent strains were used. Strains #502, #503, and #505 are clinical isolates, while strain #501 is a clinical test strain defective in the formation of hyphae. These strains were used both in their unicellular and ¢lamentous forms. Under the culture conditions described above, Candida is growing as single cells. The addition of human serum at 37‡C for 6 h was su⁄cient for the switch to the hyphal form. To maintain the di¡erent growth forms during the stimulation assay, the respective cells were heat inactivated for 20 min at 60‡C prior to their addition to blood. Spheroplast preparation was performed as described previously [23]. Brie£y, the appropriate yeast was grown in YEPD medium to late exponential phase (5U107 cells per ml), harvested, and washed twice with sterile water. The washed cells were suspended in Tris/HCl bu¡er (0.1 M Tris^HCl (pH 8.0); 5 mM dithiothreitol (DTT); 5 mM EDTA), incubated at 30‡C for 30 min, and washed with sterile water. Washed cells were then suspended in 1.2 M sorbitol containing 0.5 M phosphate bu¡er (pH 6.8), and spheroplasts were generated by treatment with zymolyase20T for 90 min at 30‡C [24].
3. Results 3.1. Various yeast genera cause extensive CD8 T lymphocyte responses in humans To investigate the human cellular immune responses against yeast, we selected a variety of yeasts genera which (i) exist in close surroundings with humans, i.e. on fruit and £owers or in milk, (ii) are used in the traditional food production like the brewer’s yeast S. cerevisiae or the ¢ssion yeast S. pombe, or (iii) cause opportunistic infections in immunocompromised individuals. Their phylogenetic relationship based on the 18S RNA is shown in Fig. 1. Antigen stimulations were carried out in human whole blood [16], allowing the quanti¢cation and characteriza-
Fig. 1. Phylogenetic relationship of the yeast genera analyzed for cellular immune responses. A neighbor-joining tree of the yeasts 18S RNA is shown. Sequences were aligned by the Clustal method, the tree was derived from the MegAlign program (DNASTAR). Percent nucleotide divergence is given below. The Sporothrix ‘species’ used for immunological analysis is not clearly de¢ned. For phylogenetic comparisons, we have arbitrarily used the 18S RNA sequence of Sporothrix schenkii.
tion of antigen-speci¢c memory CD4 and CD8 T lymphocytes by intracellular IFN-Q staining. Blood donors were ¢ve healthy European individuals, three females and two males around 30 years of age. All of them showed extensive CD8 T lymphocyte responses against most yeast genera (Fig. 2A). In contrast, the corresponding yeast-speci¢c CD4 T lymphocyte responses were signi¢cantly lower (only up to 0.5%; data not shown). The CD8 T cell frequencies against a certain yeast genus showed a great variety among the donors, ranging e.g. from 0.68 to 6.37% in case of Pichia pastoris. However, a general trend as to the distribution of the CD8 T cell responses could be observed. The highest responses were consistently detected against P. pastoris, W. californica, Sporothrix sp., Metschnikowia pulcherrima and Kluyveromyces lactis with mean frequencies above 1%. Among them there are environmental yeasts as well as the human pathogen Sporothrix sp. The lowest CD8 T cell responses were detected against the African ¢ssion yeast S. pombe (mean response below 0.1%). Interestingly, the CD8 T cell response against the largely consumed yeast S. cerevisiae, which is an important component in the production of bread and beer, was still signi¢cant with a mean of 0.6%. Altogether, the magnitude of the CD8 response was not correlated with the phylogenetic relatedness of the yeast genera. 3.2. The ¢lamentous rather than the single cell form of C. albicans activates CD8 T cell responses in blood of healthy donors C. albicans is the most frequently isolated fungal pathogen in humans [25,26]. The ability to reversibly switch between a unicellular and a ¢lamentous form is thought to be important for its virulence [27,28]. Antigen stimulations were carried out with four C. albicans strains, three were clinical isolates while the fourth was a clinical test strain restricted to the unicellular form. Using viable C. albicans yeast cells for stimulation, switching to the
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Fig. 2. Extensive human CD8 T cell responses against various yeast genera. Whole blood cells of ¢ve healthy individuals (Do1^Do5) were stimulated by addition of yeast cells from various genera (A). Yeast-speci¢c CD8 T cells were quanti¢ed as CD8-, CD69- and IFN-Q triple-positive lymphocytes by £ow cytometry. Frequencies are given as percent of total CD8 T cells. Control stimulations contain costimulatory antibodies but lack yeast cells. For C. albicans (B) three clinical isolates (strains #502, #503, and #505) and one clinical test strain defective in the formation of hyphae (strain #501) were used. The Candida strains were used both in their unicellular and ¢lamentous forms. To maintain the di¡erent growth forms during the stimulation assay, the respective cells were heat inactivated prior to their addition to blood.
¢lamentous form of the fungus was observed during the 6-h incubation time and a signi¢cant CD8 T cell activation was detected (data not shown). As recently shown by Fradin et al. (2003) this formation of Candida hyphae is not due to the presence of heparin, added to avoid blood clotting during the incubation periods [26]. To test whether the unicellular or the hyphal form induced T cell activation, the respective growth forms were arrested by heat inactivation. Regarding the single cell form, a low CD8 T cell response was only detected against C. albicans strain #502 (Do1: 0.1%; Do3 : 0.1%). The CD8 T cell frequencies against the hyphal form of all strains were high and ranged from 0.72 to 1.0% for Do1 and from
0.21 to 0.32% for Do3 respectively (Fig. 2B). The clinical test strain #501 is defective in hyphae formation and did not activate CD8 T cells (Fig. 2B). 3.3. Granulocytes play an important role in the activation of yeast-speci¢c memory CD8 T cell responses Neutrophil granulocytes and monocytes are the cell populations that take up yeast cells during the whole blood assay [29]. Neutrophils are the major leukocyte subpopulation in blood and represent an important component in the innate immune system. Their role in adaptive immune responses, however, is less well de¢ned. Since they
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were the major yeast-containing cell population and express high levels of MHC class I proteins, it was possible that they participated in the activation of the yeast-speci¢c memory CD8 T cell responses. This was con¢rmed by comparing the CD8 T cell responses induced by yeast cells in whole blood with that induced by yeast cells in PBMC of the same blood donor (Fig. 3). Blood and PBMC of two donors (Do5 and Do1, see Fig. 2) were incubated with a ¢xed number of yeast cells and the frequency of yeastspeci¢c CD8 T cells was determined by £ow cytometry. The PBMC preparations after density gradient centrifugation contained 6 5% polymorphonuclear leukocytes. The yeast cell uptake by monocytes was comparable excluding a signi¢cant alteration by the presence of complement factors in case of the whole blood assay (data not shown). The frequency of yeast-speci¢c CD8 T cells in the PBMC experiment was markedly reduced by a mean of 44% (range 27.9^65.9%) for donor 5 and 77% (range 63.9^ 86.5%) for donor 1. In contrast, stimulations using soluble antigens were not in£uenced or even enhanced by the lack of granulocytes. Thus, neutrophils seem to play an important role in the activation of the extensive CD8 T cell responses against di¡erent yeast genera. 3.4. The CD8 T cell response against yeast is MHC class I-restricted To demonstrate the MHC class I restriction of the yeast-speci¢c CD8 T cell response, a titrated amount of the pan-HLA class I antibody W6/32 was added to whole blood during the stimulation with W. californica, a yeast genus to which the highest responses had been determined (see Fig. 2). CD8 T cell activation was almost completely inhibited by W6/32 with a reduction of 93, 82 and 92% for
283
Fig. 4. The yeast-speci¢c CD8 T cell response is MHC I-restricted. Human whole blood was stimulated with W. californica as in Fig. 2. MHC-I restriction is demonstrated by the addition of anti-MHC class I or anti-HLA-B7 antibodies. Yeast-speci¢c CD8 T cells were quanti¢ed as CD8-, CD69- and IFN-Q triple-positive lymphocytes by £ow cytometry. The frequencies of yeast-speci¢c CD8 T cells of three donors as well as their HLA-B7 phenotypes are given (Do1, Do2 and Do4, arbitrarily chosen, see Fig. 2).
three di¡erent blood donors respectively (Fig. 4). Likewise, we used an inhibitory HLA-B7-speci¢c antibody and carried out the stimulation assay using blood from two HLA-B7-positive donors (Do2 and Do4, see Fig. 2) and one HLA-B7-negative donor (Do1, see Fig. 2). A partly reduced response was detected in blood of the HLA-B7-positive donors (reduction of 48 and 62% for donors 2 and 4, respectively) whereas no inhibition of the CD8 response was observed for the blood donor lacking HLA-B7 (Fig. 4). Besides these MHC class I-restricted CD8 T cells, yeast-mediated stimulation of whole blood also led to IFN-Q production in around 3% of CD33 CD16+ and/or CD56+ natural killer cells. 3.5. The yeast cell wall contains all the immunogens inducing the extensive CD8 T cell response In several studies mannoproteins have been identi¢ed as
Fig. 3. Comparative analysis of yeast-speci¢c CD8 T cell stimulation in whole blood and PBMC. Human whole blood and PBMC of the same individual were simultaneously stimulated with various yeasts. Yeastspeci¢c CD8 T cells were quanti¢ed as CD8-, CD69- and IFN-Q triplepositive lymphocytes by £ow cytometry. The yeast-speci¢c CD8 T cell frequencies of two donors (Do5 and Do1, arbitrarily chosen, see Fig. 2) are given. Cytomegalovirus antigen and Staphylococcus enterotoxin B (SEB), respectively, were used as control antigens.
Fig. 5. The immunogenic antigens are located within the yeast cell wall. Yeast spheroplasts were generated by zymolyase treatment and added to human whole blood. Yeast-speci¢c CD8 T cells were quanti¢ed as CD8CD69- and IFN-Q triple-positive lymphocytes by £ow cytometry. The frequency of yeast-speci¢c CD8 T cells of one representative blood donor is given (Do2, arbitrarily chosen, see Fig. 2).
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Fig. 6. The yeast-speci¢c CD8 T cells are end-di¡erentiated. Human whole blood was stimulated with W. californica. Yeast-speci¢c CD8 T cells were analyzed for expression of CD27 or CCR7, respectively, by £ow cytometry after staining with speci¢c antibodies. The percentage of positive and negative cells, respectively, of two donors is given (Do1 and Do2, arbitrarily chosen, see Fig. 2).
major targets of immune responses to various fungi. For the human pathogens C. neoformans or C. albicans, humoral and cellular immune responses against cell surface epitopes have been described [30,31]. To test whether the MHC class I-restricted CD8 T cell responses analyzed here were also directed against cell wall immunogens, spheroplasts of P. pastoris, Sporothrix sp. and W. californica were generated via enzymatic cell wall degradation by zymolyase treatment and used in the stimulation assay. The cell wall removal caused the disappearance of the CD8 T lymphocyte response to frequencies below 0.05% in all cases (Fig. 5), however, it did not a¡ect yeast cell uptake by APC (data not shown). Thus, the observed extensive yeast-speci¢c CD8 T cell response seems solely directed to epitopes of cell wall proteins. 3.6. Yeast-speci¢c CD8 T cells are end-di¡erentiated e¡ector memory cells The di¡erentiation status of the yeast-speci¢c CD8 T cells was determined by staining with antibodies against the maturation marker CD27 and the T lymphocyte homing molecule CCR7 in parallel. Whole blood of two donors (Do1 and Do2, see Fig. 2) was stimulated with W. californica and the yeast-speci¢c CD8 cells were quantitated and characterized by £ow cytometry. The great majority of the cells lacked both CD27 and CCR7 expression, i.e. of the yeast-speci¢c CD8 T cells about 90% were negative for CD27 and about 77% were negative for CCR7 (Fig. 6). Such a phenotype is typical for end-di¡erentiated e¡ector memory T cells.
4. Discussion In this study an analysis of the frequencies and phenotypic characteristics of yeast-speci¢c T cell responses in
humans was performed. Strikingly, all ¢ve healthy volunteers tested showed equally extensive MHC class I-restricted CD8 T cell responses against several yeast genera. In contrast, yeast-speci¢c CD4 T cells were of low frequency or could barely be detected. The analysis of the expression of the T cell di¡erentiation markers CD27 and CCR7 revealed an end-di¡erentiated phenotype of the IFN-Q-producing e¡ector CD8 T cells [21,22]. However, the speci¢c responses against the 14 yeast genera tested were highly variable and di¡ered in speci¢city between the volunteers. For example, the highest responses with mean frequencies above 1% were detected against P. pastoris, W. californica, Sporothrix sp., M. pulcherrima and K. lactis while the lowest frequencies were seen against the African ¢ssion yeast S. pombe (mean response below 0.1%). Taking e.g. P. pastoris, a wide range from 0.68 to 6.37% speci¢c CD8 T cell frequencies was observed among the responders. Interestingly, the CD8 T cell response against the commonly consumed baker’s and brewer’s yeast S. cerevisiae was still signi¢cant with a mean of 0.6%. An exceptional position takes the pathogenic yeast C. albicans because it is able to reversibly switch between a unicellular and a ¢lamentous form, both of which can be found in infected tissues [32]. The capacity to switch between these forms seems to be important for the virulence, however, it is still an open question which of the growth forms is responsible for it [27]. In this study we demonstrate a di¡erence in their capacity to activate Candidaspeci¢c CD8 T cells. A consistent and signi¢cant CD8 T cell response was only detectable against the Candida hyphae. This contrasts observations made in mice where only the unicellular form of C. albicans is able to mount a Th1-type, protective immunity [33]. If one sums up the yeast-speci¢c CD8 T cell frequencies for the highest responder one might predict that around 25% of all his CD8 T cells are yeast-speci¢c (see Fig. 2). Most likely this assumed frequency would further increase if one would measure additional yeast species that have not yet been included in our study. Such an extremely high frequency of an antigen-speci¢c CD8 T cell response is only known for acute viral infections when the adaptive immune response has to rapidly control the exponential expansion of a virus [34]. However, within a healthy individual, the frequency of memory CD8 responses is usually an order of magnitude lower. Given the similarity of the yeast cell wall components and their immunodominance [31,35,36], it seems likely that part of the measured CD8 responses are directed against cross-reactive epitopes. This would ¢t well with the promiscuous recognition potential of a TCR [37] and, from an evolutionary point of view, would help to maintain memory and mount a rapid immune response against a whole class of microorganisms [38]. At this stage, however, it is not possible to demonstrate cross-reactivity at the epitope level because neither the HLA class I-restricted immunogens, nor the primary
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structures of the cell wall proteins in non-Saccharomyces yeast genera are known. An alternative explanation for a high T cell response against yeasts could be the presence of a superantigen. Superantigens like Staphylococcus enterotoxins crosslink MHC class II proteins on antigen presenting cells with speci¢c VL regions on the TCR of T cells and thereby stimulate both CD4 and CD8 T cells [39]. However, the yeast-speci¢c T cell response reported here was highly biased towards CD8 T cells. Furthermore, partial analysis of the VL-TCR regions of some of the yeast-responsive CD8 cells revealed a discordance between the blood donors again arguing against a superantigen e¡ect (data not shown). The peculiarities of some threatening virus infections like those with HIV and HCV have illustrated the need for vaccines that stimulate virus-speci¢c CD8-positive CTL [40^43]. The work presented here would suggest that yeast might be particularly suited for inducing such responses in humans. As an antigen carrier, yeast would have several advantages. Firstly, it combines the advantage of a single cell organism (i.e. small size and ease of cultivation) with that of an eukaryotic cell capable to perform N-glycosylation and complex posttranslational modi¢cations [44,45]. Secondly, yeast could be orally applied following its natural entry into the human body. This would induce mucosal immunity which is highly desirable in case of viruses such as HIV. Thirdly, yeast has been shown to be e⁄ciently phagocytosed by dendritic cells and exerted a strong adjuvant e¡ect with increased MHC-restricted epitope presentation to T cells [46^48]. Finally, whole recombinant S. cerevisiae cells expressing tumor antigens were recently shown to induce antigen-speci¢c T cell responses that mediated e⁄cient tumor protection in vaccinated animals [49]. In summary, our presented ¢ndings indicate that yeast not only is an attractive model organism in studying eukaryotic cell biology, but it is also becoming increasingly interesting with respect to its potential for the development of e¡ective novel CTL vaccines.
Acknowledgements We thank Simone Martens for excellent assistance and Nikolaus Mu«ller-Lantzsch for continuous support. This work was supported by the Deutsche Forschungsgemeinschaft and a grant from the Ministerium fu«r Bildung, Kultur und Wissenschaft of the Saarland (E-TTT) to F.B. and M.J.S.
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Extensive MHC class I-restricted CD8 T lymphocyte responses against various yeast genera in humans Tanja Heintel a
a;1
, Frank Breinig
b;1
, Manfred J. Schmitt b , Andreas Meyerhans
a;
Department of Virology, Institute of Medical Microbiology and Hygiene, Building 47, University of the Saarland, 66421 Homburg, Germany b Department of Applied Molecular Biology, University of the Saarland, 66123 Saarbru«cken, Germany Received 9 May 2003; received in revised form 20 August 2003; accepted 13 September 2003 First published online 21 October 2003
Abstract The human cellular immune response against 14 distantly related yeast species was analyzed by intracellular cytokine staining of lymphocytes after ex vivo stimulation of whole blood. While the CD4 T cell response was marginal, extensive MHC class I-restricted CD8 T cell responses were detected against a number of species including spoiling, environmental and human pathogenic yeasts. The yeastspecific CD8 T cells expressed interferon-Q but lacked expression of CD27 and CCR7, indicating that they were end-differentiated effector memory cells. Mainly intact yeast cells rather than spheroplasts were able to induce cytokine expression in T cells demonstrating that the dominant immunogens were located in the yeast cell wall. Together these data underline the importance of the cellular immune response in protecting humans against yeast and fungal infections. And, from another perspective, recombinant yeast suggests itself as a potential vaccine candidate to efficiently induce antigen-specific CD8 T cell responses. 5 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords : Human ; Fungal infection ; Cellular activation; T lymphocyte
1. Introduction Yeast species are unicellular fungi, most of them classi¢ed within the ascomycetes. They usually £ourish in habitats like fruits, £owers and the bark of trees and thus exist in the close surroundings of humans. In particular Saccharomyces cerevisiae, which is known as baker’s or brewer’s yeast, has been used for centuries for the production of bread and alcoholic beverages. Likewise, Schizosaccharomyces pombe is used for brewing of millet beer in Africa. A number of yeast species live in symbiosis with animals and pathogenicity in humans is rare. However, individuals with an impaired immune system due to immunosuppressive drugs or infections caused by agents including human immunode¢ciency virus (HIV), may experience life-threatening systemic infections with pathogenic strains of Cryptococcus neoformans and Candida albicans [1^4].
* Corresponding author. Tel. : +49 (6841) 162 3990 ; Fax : +49 (6841) 162 3980. E-mail address : [email protected] (A. Meyerhans). 1
These authors contributed equally to this work.
Both innate and acquired immune responses participate in the protection against yeast infections. Polymorphonuclear neutrophils were shown to play a critical role in providing early resistance against the human pathogenic yeasts C. neoformans and Paracoccidioides brasiliensis [5,6]. In untreated HIV-infected patients with advanced immunode¢ciency, such leukocytes have diminished antimicrobial and secretory functions which result in a defect in protection against C. neoformans and other opportunistic pathogens [3,7,8]. For the two major human pathogenic yeasts, C. neoformans and C. albicans, protective as well as non-protective antibodies have been identi¢ed, suggesting that the e⁄cacy of antibody-mediated protection is a function of isotype, dose and speci¢city [9,10]. Adoptive lymphocyte transfer experiments in mice have shown that cellular immunity can also mediate protection against several yeast species while defects in cellular immune responses enhance the susceptibility towards such infections [11,12]. Regarding the T helper (Th) phenotype, Th1 cells usually result in resistance while Th2 cell responses are associated with susceptibility to C. albicans and C. neoformans infections [12^14]. Until today, most experiments on cellular immune responses against yeast refer to human pathogenic yeast
0928-8244 / 03 / $22.00 5 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0928-8244(03)00294-3
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species. To globally characterize the human T cell response against yeast, we have investigated the stimulation of T lymphocytes by a variety of distantly related yeast genera from di¡erent habitats, including spoiling, environmental, opportunistically pathogenic as well as traditionally cultured yeasts. An ex vivo whole blood assay was performed that permits the identi¢cation and simultaneous quanti¢cation of antigen-speci¢c CD4 and CD8 T cells as already described for HIV- and CMV-speci¢c T cells [15^ 17]. Whole blood is incubated for 6 h with the respective antigens. These are taken up by antigen presenting cells within the blood and subsequently enter the pathways of antigen processing and presentation [18]. Interestingly, the processed peptides are presented by major histocompatibility proteins of classes I and II, thus allowing to measure MHC class I-dependent CD8 T cell activation and MHC class II-dependent CD4 T cell activation. The speci¢c T cell stimulation then results in the upregulation of the early activation marker CD69 and the production of interferon-Q (IFN-Q) and other cytokines which can be quanti¢ed by £ow cytometry [16,18]. During the 6-h incubation time used here, only e¡ector and memory CD4 and CD8 T cells are speci¢cally stimulated, whereas the di¡erentiation kinetics of naive T cells do not allow such a rapid activation [19,20]. Therefore the whole blood assay under our conditions is able to quantify pre-existing cellular immune responses. Here we show that an e⁄cient uptake of intact yeast cells by phagocytosis is followed by an extensive yeastspeci¢c and MHC class I-restricted immune response of CD8 T lymphocytes. The immunogens inducing the CD8 T cell responses are located within the yeast cell wall. Moreover, these yeast-speci¢c CD8 T cells are characterized by an end-di¡erentiated e¡ector memory phenotype, because they show the typical lack of the di¡erentiation markers CD27 and CCR7 [21,22]. Together these data underline the potential of recombinant yeast in the development of novel vaccines inducing HLA class I-restricted CD8 T cell responses.
2. Materials and methods 2.1. Stimulation of yeast-speci¢c CD4 and CD8 T cells within whole blood Antigen stimulation of yeast-speci¢c T cells was performed in whole blood as described previously [16]. Blood donors were ¢ve healthy European individuals, three females and two males around 30 years of age. For some experiments blood from arbitrarily chosen donors among these was used. Brie£y, 450 Wl of heparinized blood were mixed with 1 Wg ml31 of the costimulatory antibodies KCD28 and KCD49d each (clones L293 and L25.3, BD Pharmingen, Heidelberg, Germany). Stimulations were carried out using titrated amounts of yeast cells
(7.5U105 cells per 450 Wl blood). To obtain optimal T cell responses, titration of the yeast cell amount was performed with 7.5U102 to 7.5U106 cells of the highly immunogenic yeast Williopsis californica (not shown). As negative control, cells were stimulated with costimulatory antibodies only. The whole blood was incubated in 15-ml polypropylene tubes at 37‡C/5% CO2 for a total of 6 h. During the last 4 h 10 Wg ml31 of Brefeldin A (Sigma, Deisenhofen, Germany) was added to inhibit the cytokine secretion. After incubation of the blood with 2 mM ethylenediamine tetraacetic acid (EDTA) for 15 min, erythrocytes were lysed and leukocytes were ¢xed for 10 min using Becton Dickinson lysing solution according to the manufacturer’s instruction. Thereafter, cells were washed once with FACS bu¡er (phosphate-bu¡ered saline (PBS), 5% ¢ltered fetal calf serum (FCS), 0.5% bovine serum albumin (BSA), 0.07% NaN3 ) and left overnight at 4‡C. For the determination of the frequency of antigen-speci¢c CD4 and CD8 T cells by £ow cytometry, the ¢xed leukocytes were permeabilized with 2 ml FACS bu¡er containing 0.1% saponin (Sigma) for 10 min at room temperature (RT). The immunostaining was carried out for 30 min at RT in the dark using saturating conditions of the antibodies anti-CD4 and anti-CD8 (clone SK3 and clone SK1, PerCP, Becton Dickinson, Heidelberg, Germany), anti-IFN-Q (clone 4S.B3, £uorescein isothiocyanate (FITC) or phenylethylamine (PE), BD Pharmingen) or anti-CD69 (clone TP1.55.3, PE, Beckman Coulter, Krefeld, Germany). For a further characterization of yeastspeci¢c T cells, the antibodies anti-CD27 (clone M-T271, FITC, BD Pharmingen), anti-CCR7 (clone 3D12, Max Delbru«ck Centrum) and donkey anti-rat (PE, Dianova, Hamburg, Germany) were used. Cells were washed once with FACS bu¡er and ¢xed using 1% paraformaldehyde. CD4- and CD8-positive T cells were identi¢ed by gating on the lymphocyte population via cell size and granularity and their high CD4 and CD8 expression level, respectively. At least 25 000 CD4- and CD8-positive lymphocytes were analyzed on a FACScan (Becton Dickinson) using the Cellquest Software. Speci¢cally activated T lymphocytes were identi¢ed and quanti¢ed as CD69 and IFN-Q double-positive cells. The addition of costimulatory antibodies only (negative control) led not to a signi¢cant T cell stimulation (frequencies below 0.05%). 2.2. Yeast-speci¢c stimulation of peripheral blood mononuclear cells (PBMC) Human PBMC were isolated from fresh, heparinized blood through density gradient centrifugation over Ficoll-Hypaque (Pharmacia, Freiburg, Germany). Antigen stimulations were carried out in polypropylene FACS tubes using 2U106 cells in 400 Wl RPMI+5% human serum (Sigma) per sample. Costimulatory antibodies and yeast cells were added as mentioned above. After an incubation period of 30 min at 37‡C/5% CO2 , the samples were cen-
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trifuged for 5 min at 1100 rpm and incubated for another 90 min. This centrifugation step was repeated after the subsequent addition of Brefeldin A. The cells were ¢rst incubated with 2 mM EDTA for 15 min (see above), washed with 2 ml PBS+0.02% EDTA and ¢xed with 4% paraformaldehyde for 5 min at RT. Fixation was stopped by the addition of FACS bu¡er. 2.3. Yeast genera, yeast culture and spheroplast preparation The various yeast genera used in this study were provided by the yeast strain collection of the Institute of Applied Molecular Biology, Saarland University. All yeast strains were grown in YEPD medium (2% glucose, 2% peptone, 1% yeast extract) at 30‡C. Before adding to the whole blood (see above), yeasts were washed three times in PBS (5000 rpm, 10 min, RT). For C. albicans, four di¡erent strains were used. Strains #502, #503, and #505 are clinical isolates, while strain #501 is a clinical test strain defective in the formation of hyphae. These strains were used both in their unicellular and ¢lamentous forms. Under the culture conditions described above, Candida is growing as single cells. The addition of human serum at 37‡C for 6 h was su⁄cient for the switch to the hyphal form. To maintain the di¡erent growth forms during the stimulation assay, the respective cells were heat inactivated for 20 min at 60‡C prior to their addition to blood. Spheroplast preparation was performed as described previously [23]. Brie£y, the appropriate yeast was grown in YEPD medium to late exponential phase (5U107 cells per ml), harvested, and washed twice with sterile water. The washed cells were suspended in Tris/HCl bu¡er (0.1 M Tris^HCl (pH 8.0); 5 mM dithiothreitol (DTT); 5 mM EDTA), incubated at 30‡C for 30 min, and washed with sterile water. Washed cells were then suspended in 1.2 M sorbitol containing 0.5 M phosphate bu¡er (pH 6.8), and spheroplasts were generated by treatment with zymolyase20T for 90 min at 30‡C [24].
3. Results 3.1. Various yeast genera cause extensive CD8 T lymphocyte responses in humans To investigate the human cellular immune responses against yeast, we selected a variety of yeasts genera which (i) exist in close surroundings with humans, i.e. on fruit and £owers or in milk, (ii) are used in the traditional food production like the brewer’s yeast S. cerevisiae or the ¢ssion yeast S. pombe, or (iii) cause opportunistic infections in immunocompromised individuals. Their phylogenetic relationship based on the 18S RNA is shown in Fig. 1. Antigen stimulations were carried out in human whole blood [16], allowing the quanti¢cation and characteriza-
Fig. 1. Phylogenetic relationship of the yeast genera analyzed for cellular immune responses. A neighbor-joining tree of the yeasts 18S RNA is shown. Sequences were aligned by the Clustal method, the tree was derived from the MegAlign program (DNASTAR). Percent nucleotide divergence is given below. The Sporothrix ‘species’ used for immunological analysis is not clearly de¢ned. For phylogenetic comparisons, we have arbitrarily used the 18S RNA sequence of Sporothrix schenkii.
tion of antigen-speci¢c memory CD4 and CD8 T lymphocytes by intracellular IFN-Q staining. Blood donors were ¢ve healthy European individuals, three females and two males around 30 years of age. All of them showed extensive CD8 T lymphocyte responses against most yeast genera (Fig. 2A). In contrast, the corresponding yeast-speci¢c CD4 T lymphocyte responses were signi¢cantly lower (only up to 0.5%; data not shown). The CD8 T cell frequencies against a certain yeast genus showed a great variety among the donors, ranging e.g. from 0.68 to 6.37% in case of Pichia pastoris. However, a general trend as to the distribution of the CD8 T cell responses could be observed. The highest responses were consistently detected against P. pastoris, W. californica, Sporothrix sp., Metschnikowia pulcherrima and Kluyveromyces lactis with mean frequencies above 1%. Among them there are environmental yeasts as well as the human pathogen Sporothrix sp. The lowest CD8 T cell responses were detected against the African ¢ssion yeast S. pombe (mean response below 0.1%). Interestingly, the CD8 T cell response against the largely consumed yeast S. cerevisiae, which is an important component in the production of bread and beer, was still signi¢cant with a mean of 0.6%. Altogether, the magnitude of the CD8 response was not correlated with the phylogenetic relatedness of the yeast genera. 3.2. The ¢lamentous rather than the single cell form of C. albicans activates CD8 T cell responses in blood of healthy donors C. albicans is the most frequently isolated fungal pathogen in humans [25,26]. The ability to reversibly switch between a unicellular and a ¢lamentous form is thought to be important for its virulence [27,28]. Antigen stimulations were carried out with four C. albicans strains, three were clinical isolates while the fourth was a clinical test strain restricted to the unicellular form. Using viable C. albicans yeast cells for stimulation, switching to the
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Fig. 2. Extensive human CD8 T cell responses against various yeast genera. Whole blood cells of ¢ve healthy individuals (Do1^Do5) were stimulated by addition of yeast cells from various genera (A). Yeast-speci¢c CD8 T cells were quanti¢ed as CD8-, CD69- and IFN-Q triple-positive lymphocytes by £ow cytometry. Frequencies are given as percent of total CD8 T cells. Control stimulations contain costimulatory antibodies but lack yeast cells. For C. albicans (B) three clinical isolates (strains #502, #503, and #505) and one clinical test strain defective in the formation of hyphae (strain #501) were used. The Candida strains were used both in their unicellular and ¢lamentous forms. To maintain the di¡erent growth forms during the stimulation assay, the respective cells were heat inactivated prior to their addition to blood.
¢lamentous form of the fungus was observed during the 6-h incubation time and a signi¢cant CD8 T cell activation was detected (data not shown). As recently shown by Fradin et al. (2003) this formation of Candida hyphae is not due to the presence of heparin, added to avoid blood clotting during the incubation periods [26]. To test whether the unicellular or the hyphal form induced T cell activation, the respective growth forms were arrested by heat inactivation. Regarding the single cell form, a low CD8 T cell response was only detected against C. albicans strain #502 (Do1: 0.1%; Do3 : 0.1%). The CD8 T cell frequencies against the hyphal form of all strains were high and ranged from 0.72 to 1.0% for Do1 and from
0.21 to 0.32% for Do3 respectively (Fig. 2B). The clinical test strain #501 is defective in hyphae formation and did not activate CD8 T cells (Fig. 2B). 3.3. Granulocytes play an important role in the activation of yeast-speci¢c memory CD8 T cell responses Neutrophil granulocytes and monocytes are the cell populations that take up yeast cells during the whole blood assay [29]. Neutrophils are the major leukocyte subpopulation in blood and represent an important component in the innate immune system. Their role in adaptive immune responses, however, is less well de¢ned. Since they
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were the major yeast-containing cell population and express high levels of MHC class I proteins, it was possible that they participated in the activation of the yeast-speci¢c memory CD8 T cell responses. This was con¢rmed by comparing the CD8 T cell responses induced by yeast cells in whole blood with that induced by yeast cells in PBMC of the same blood donor (Fig. 3). Blood and PBMC of two donors (Do5 and Do1, see Fig. 2) were incubated with a ¢xed number of yeast cells and the frequency of yeastspeci¢c CD8 T cells was determined by £ow cytometry. The PBMC preparations after density gradient centrifugation contained 6 5% polymorphonuclear leukocytes. The yeast cell uptake by monocytes was comparable excluding a signi¢cant alteration by the presence of complement factors in case of the whole blood assay (data not shown). The frequency of yeast-speci¢c CD8 T cells in the PBMC experiment was markedly reduced by a mean of 44% (range 27.9^65.9%) for donor 5 and 77% (range 63.9^ 86.5%) for donor 1. In contrast, stimulations using soluble antigens were not in£uenced or even enhanced by the lack of granulocytes. Thus, neutrophils seem to play an important role in the activation of the extensive CD8 T cell responses against di¡erent yeast genera. 3.4. The CD8 T cell response against yeast is MHC class I-restricted To demonstrate the MHC class I restriction of the yeast-speci¢c CD8 T cell response, a titrated amount of the pan-HLA class I antibody W6/32 was added to whole blood during the stimulation with W. californica, a yeast genus to which the highest responses had been determined (see Fig. 2). CD8 T cell activation was almost completely inhibited by W6/32 with a reduction of 93, 82 and 92% for
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Fig. 4. The yeast-speci¢c CD8 T cell response is MHC I-restricted. Human whole blood was stimulated with W. californica as in Fig. 2. MHC-I restriction is demonstrated by the addition of anti-MHC class I or anti-HLA-B7 antibodies. Yeast-speci¢c CD8 T cells were quanti¢ed as CD8-, CD69- and IFN-Q triple-positive lymphocytes by £ow cytometry. The frequencies of yeast-speci¢c CD8 T cells of three donors as well as their HLA-B7 phenotypes are given (Do1, Do2 and Do4, arbitrarily chosen, see Fig. 2).
three di¡erent blood donors respectively (Fig. 4). Likewise, we used an inhibitory HLA-B7-speci¢c antibody and carried out the stimulation assay using blood from two HLA-B7-positive donors (Do2 and Do4, see Fig. 2) and one HLA-B7-negative donor (Do1, see Fig. 2). A partly reduced response was detected in blood of the HLA-B7-positive donors (reduction of 48 and 62% for donors 2 and 4, respectively) whereas no inhibition of the CD8 response was observed for the blood donor lacking HLA-B7 (Fig. 4). Besides these MHC class I-restricted CD8 T cells, yeast-mediated stimulation of whole blood also led to IFN-Q production in around 3% of CD33 CD16+ and/or CD56+ natural killer cells. 3.5. The yeast cell wall contains all the immunogens inducing the extensive CD8 T cell response In several studies mannoproteins have been identi¢ed as
Fig. 3. Comparative analysis of yeast-speci¢c CD8 T cell stimulation in whole blood and PBMC. Human whole blood and PBMC of the same individual were simultaneously stimulated with various yeasts. Yeastspeci¢c CD8 T cells were quanti¢ed as CD8-, CD69- and IFN-Q triplepositive lymphocytes by £ow cytometry. The yeast-speci¢c CD8 T cell frequencies of two donors (Do5 and Do1, arbitrarily chosen, see Fig. 2) are given. Cytomegalovirus antigen and Staphylococcus enterotoxin B (SEB), respectively, were used as control antigens.
Fig. 5. The immunogenic antigens are located within the yeast cell wall. Yeast spheroplasts were generated by zymolyase treatment and added to human whole blood. Yeast-speci¢c CD8 T cells were quanti¢ed as CD8CD69- and IFN-Q triple-positive lymphocytes by £ow cytometry. The frequency of yeast-speci¢c CD8 T cells of one representative blood donor is given (Do2, arbitrarily chosen, see Fig. 2).
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Fig. 6. The yeast-speci¢c CD8 T cells are end-di¡erentiated. Human whole blood was stimulated with W. californica. Yeast-speci¢c CD8 T cells were analyzed for expression of CD27 or CCR7, respectively, by £ow cytometry after staining with speci¢c antibodies. The percentage of positive and negative cells, respectively, of two donors is given (Do1 and Do2, arbitrarily chosen, see Fig. 2).
major targets of immune responses to various fungi. For the human pathogens C. neoformans or C. albicans, humoral and cellular immune responses against cell surface epitopes have been described [30,31]. To test whether the MHC class I-restricted CD8 T cell responses analyzed here were also directed against cell wall immunogens, spheroplasts of P. pastoris, Sporothrix sp. and W. californica were generated via enzymatic cell wall degradation by zymolyase treatment and used in the stimulation assay. The cell wall removal caused the disappearance of the CD8 T lymphocyte response to frequencies below 0.05% in all cases (Fig. 5), however, it did not a¡ect yeast cell uptake by APC (data not shown). Thus, the observed extensive yeast-speci¢c CD8 T cell response seems solely directed to epitopes of cell wall proteins. 3.6. Yeast-speci¢c CD8 T cells are end-di¡erentiated e¡ector memory cells The di¡erentiation status of the yeast-speci¢c CD8 T cells was determined by staining with antibodies against the maturation marker CD27 and the T lymphocyte homing molecule CCR7 in parallel. Whole blood of two donors (Do1 and Do2, see Fig. 2) was stimulated with W. californica and the yeast-speci¢c CD8 cells were quantitated and characterized by £ow cytometry. The great majority of the cells lacked both CD27 and CCR7 expression, i.e. of the yeast-speci¢c CD8 T cells about 90% were negative for CD27 and about 77% were negative for CCR7 (Fig. 6). Such a phenotype is typical for end-di¡erentiated e¡ector memory T cells.
4. Discussion In this study an analysis of the frequencies and phenotypic characteristics of yeast-speci¢c T cell responses in
humans was performed. Strikingly, all ¢ve healthy volunteers tested showed equally extensive MHC class I-restricted CD8 T cell responses against several yeast genera. In contrast, yeast-speci¢c CD4 T cells were of low frequency or could barely be detected. The analysis of the expression of the T cell di¡erentiation markers CD27 and CCR7 revealed an end-di¡erentiated phenotype of the IFN-Q-producing e¡ector CD8 T cells [21,22]. However, the speci¢c responses against the 14 yeast genera tested were highly variable and di¡ered in speci¢city between the volunteers. For example, the highest responses with mean frequencies above 1% were detected against P. pastoris, W. californica, Sporothrix sp., M. pulcherrima and K. lactis while the lowest frequencies were seen against the African ¢ssion yeast S. pombe (mean response below 0.1%). Taking e.g. P. pastoris, a wide range from 0.68 to 6.37% speci¢c CD8 T cell frequencies was observed among the responders. Interestingly, the CD8 T cell response against the commonly consumed baker’s and brewer’s yeast S. cerevisiae was still signi¢cant with a mean of 0.6%. An exceptional position takes the pathogenic yeast C. albicans because it is able to reversibly switch between a unicellular and a ¢lamentous form, both of which can be found in infected tissues [32]. The capacity to switch between these forms seems to be important for the virulence, however, it is still an open question which of the growth forms is responsible for it [27]. In this study we demonstrate a di¡erence in their capacity to activate Candidaspeci¢c CD8 T cells. A consistent and signi¢cant CD8 T cell response was only detectable against the Candida hyphae. This contrasts observations made in mice where only the unicellular form of C. albicans is able to mount a Th1-type, protective immunity [33]. If one sums up the yeast-speci¢c CD8 T cell frequencies for the highest responder one might predict that around 25% of all his CD8 T cells are yeast-speci¢c (see Fig. 2). Most likely this assumed frequency would further increase if one would measure additional yeast species that have not yet been included in our study. Such an extremely high frequency of an antigen-speci¢c CD8 T cell response is only known for acute viral infections when the adaptive immune response has to rapidly control the exponential expansion of a virus [34]. However, within a healthy individual, the frequency of memory CD8 responses is usually an order of magnitude lower. Given the similarity of the yeast cell wall components and their immunodominance [31,35,36], it seems likely that part of the measured CD8 responses are directed against cross-reactive epitopes. This would ¢t well with the promiscuous recognition potential of a TCR [37] and, from an evolutionary point of view, would help to maintain memory and mount a rapid immune response against a whole class of microorganisms [38]. At this stage, however, it is not possible to demonstrate cross-reactivity at the epitope level because neither the HLA class I-restricted immunogens, nor the primary
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structures of the cell wall proteins in non-Saccharomyces yeast genera are known. An alternative explanation for a high T cell response against yeasts could be the presence of a superantigen. Superantigens like Staphylococcus enterotoxins crosslink MHC class II proteins on antigen presenting cells with speci¢c VL regions on the TCR of T cells and thereby stimulate both CD4 and CD8 T cells [39]. However, the yeast-speci¢c T cell response reported here was highly biased towards CD8 T cells. Furthermore, partial analysis of the VL-TCR regions of some of the yeast-responsive CD8 cells revealed a discordance between the blood donors again arguing against a superantigen e¡ect (data not shown). The peculiarities of some threatening virus infections like those with HIV and HCV have illustrated the need for vaccines that stimulate virus-speci¢c CD8-positive CTL [40^43]. The work presented here would suggest that yeast might be particularly suited for inducing such responses in humans. As an antigen carrier, yeast would have several advantages. Firstly, it combines the advantage of a single cell organism (i.e. small size and ease of cultivation) with that of an eukaryotic cell capable to perform N-glycosylation and complex posttranslational modi¢cations [44,45]. Secondly, yeast could be orally applied following its natural entry into the human body. This would induce mucosal immunity which is highly desirable in case of viruses such as HIV. Thirdly, yeast has been shown to be e⁄ciently phagocytosed by dendritic cells and exerted a strong adjuvant e¡ect with increased MHC-restricted epitope presentation to T cells [46^48]. Finally, whole recombinant S. cerevisiae cells expressing tumor antigens were recently shown to induce antigen-speci¢c T cell responses that mediated e⁄cient tumor protection in vaccinated animals [49]. In summary, our presented ¢ndings indicate that yeast not only is an attractive model organism in studying eukaryotic cell biology, but it is also becoming increasingly interesting with respect to its potential for the development of e¡ective novel CTL vaccines.
Acknowledgements We thank Simone Martens for excellent assistance and Nikolaus Mu«ller-Lantzsch for continuous support. This work was supported by the Deutsche Forschungsgemeinschaft and a grant from the Ministerium fu«r Bildung, Kultur und Wissenschaft of the Saarland (E-TTT) to F.B. and M.J.S.
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