Direct Binding of the pH-Regulated Protein 1 (Pra1) from Candida albicans Inhibits Cytokine Secretion by Mouse CD4+ T Cells

Opportunistic infections with the saprophytic yeast Candida albicans are a major cause of morbidity in immunocompromised patients. While the interaction of cells and molecules of innate immunity with C. albicans has been studied to great depth, comparatively little is known about the modulation of adaptive immunity by C. albicans. In particular, direct interaction of proteins secreted by C. albicans with CD4+ T cells has not been studied in detail. In a first screening approach, we identified the pH-regulated antigen 1 (Pra1) as a molecule capable of directly binding to mouse CD4+ T cells in vitro. Binding of Pra1 to the T cell surface was enhanced by extracellular Zn2+ ions which Pra1 is known to scavenge from the host in order to supply the fungus with Zn2+. In vitro stimulation assays using highly purified mouse CD4+ T cells showed that Pra1 increased proliferation of CD4+ T cells in the presence of plate-bound anti-CD3 monoclonal antibody. In contrast, secretion of effector cytokines such as IFNγ and TNF by CD4+ T cells upon anti-CD3/ anti-CD28 mAb as well as cognate antigen stimulation was reduced in the presence of Pra1. By secreting Pra1 C. albicans, thus, directly modulates and partially controls CD4+ T cell responses as shown in our in vitro assays.


INTRODUCTION
Candida albicans is a commensal on human skin and mucosal surfaces. In situations of immunosuppression, C. albicans may, however, become pathogenic. Prominent examples of C. albicans-induced pathologies are mucosal or skin candidiasis as well as C. albicans septicemia in ICU and/ or HIV/Aids patients (Klein et al., 1984;Sangeorzan et al., 1994;Leroy et al., 2009). In the latter cohorts, loss of CD4 + T cells is the hallmark of immunodeficiency. This highlights the importance of CD4 + T cells for controlling C. albicans infections in humans.
To allow commensalism, C. albicans has evolved a number of evasion strategies to protect itself from attack by the host's immune system (Zipfel et al., 2011). Immune evasion might be beneficial during commensal growth as it avoids potentially harmful inflammation and adaptive immune responses. The very same mechanisms might, however, contribute to C. albicans pathogenicity once epithelial barriers are disturbed. Research into the factors driving C. albicans pathogenicity led to the discovery of the pH-regulated antigen 1 (Pra1) as a multifaceted immune evasion protein (Zipfel et al., 2011). Pra1 interferes with innate immunity including the complement cascade on different levels thereby efficiently protecting the fungus from complement attack. Moreover, Pra1 scavenges zinc from the host, thus, ensuring sufficient supply of the fungus with this bivalent cation (Citiulo et al., 2012). For both functions, complement inhibition and zinc scavenging, Pra1 is first secreted, interacts with complement proteins or zinc in solution and then the complex of Pra1 and its binding partner are recruited back to the C. albicans surface (Zipfel et al., 2011;Citiulo et al., 2012).
As Pra1 is secreted by C. albicans we hypothesized that this fungal protein might also be capable of bypassing fungal sensing by DCs (Romani, 2011) and of directly interacting with CD4 + T cells, thus, modulating T cell function in its favor. Having established that recombinantly expressed Pra1 binds to mouse CD4 + T cells, we, thus, analyzed the impact of Pra1 on T cell activation, expansion and effector cytokine secretion. Our data suggest that C. albicans directly modulates anti-fungal immunity through secreting T cell-binding proteins like Pra1.

Mice
Wild-type C57BL/6J mice and OT-II C57BL/6J mice (Barnden et al., 1998) were bred in the animal facility of the Institute for Virology and Immunobiology, University of Würzburg. CD55 −/− C57BL/6 mice (Sun et al., 1999) were obtained from the University of Cardiff and also bred in our animal facility. Crry −/− C57BL/6 (Ruseva et al., 2009) and CD59a −/− C57BL/6 mice (Holt et al., 2001) were bred at Cardiff University. All mice were kept in a specified pathogen free conventionally housed environment and used for experiments between six and 21 weeks of age.
For staining of Pra1 a polyclonal antibody was raised in rabbits by immunization with purified recombinant Pra1. Aspf2antiserum was generated by immunization of mice with purified recombinant Aspf2. Secondary polyclonal antibodies for staining of primary antibodies were goat anti-mouse-Ig FITC and donkey anti-rabbit-Ig PE (Jackson Immunoresearch, West Grove, PA, USA). Flow cytometry was performed on a FACSCalibur or LSR II flow cytometer using either CellQuest or DIVA software (BD Bioscience, Franklin Lakes, NJ, USA). We used FlowJo (TreeStar) to further analyze FACS data.

Protein Expression and Purification
Recombinant Pra1wt and Aspf2 were expressed in Pichia pastoris and isolated via the His-tag (Luo et al., 2009;Bacher et al., 2014). For protein overexpression and purification, the pra1 gene encoding a protein lacking the C-terminal 61-amino acid was amplified from the pPICZαB-Pra1wt clone using the sequence specific forward primers ACTGAATTCTGTGGAGCCATCCGCAGTTTGAAAAAAGCG CGGCACCAGTTACGGTTACC and reverse primer ACTGGT ACCGCGCACCCTTCGCCGGGAATTG, containing the restriction sites EcoRI and KpnI. The PCR product and plasmid pPICZαB were enzymatically digested, ligated, and sub-cloned into pPICZαB (Invitrogen, Karlsruhe, Germany). The resulting plasmid pPICZαB-Pra1 C61 was transfected and overexpressed in Pichia pastoris X33 (EasySelect TM Pichia Expression Kit, Invitrogen, Karlsruhe, Germany). The Pra1 C61 was purified as described (Luo et al., 2009).

Organ Processing and FACS Stainings
Single cell suspensions were generated by mashing cervical, axillary, inguinal and mesenteric lymph nodes or spleens through a cell strainer (Falcon, Pittsburg, PA, USA). Single cell suspensions of splenocytes were then subjected to red cell lysis by hypoosmotic shock. Lymph node and red cell-lysed spleen cells were then resuspended in buffered salt solution (BSS) containing 0.1% (w/v) bovine serum albumin (BSA). Total lymph node or spleen cells were incubated with Pra1 (10 µg/ ml) or Aspf2 (10 µg/ ml) in PBS at 37 • C for 30 or 45 min. For investigation of the influence of zinc on Pra1 binding, ZnCl 2 (1, 10, or 100 µM) was added while incubating cells together with Pra1. After washing bound Pra1 or Aspf2 were detected with a polyclonal anti-Pra1-(rabbit) or anti-Aspf2-(mouse) antiserum followed by PE anti-rabbit Ig polyclonal antibody (donkey; Dianova) or FITC anti-rabbit Ig polyclonal antibody (donkey; Dianova). For further stainings the samples were blocked with normal rabbit serum (1:500) or normal mouse Ig (20 µg/ ml, Sigma) followed by incubation with anti-CD4 mAb (Alexa Fluor 647) alone or together with anti-CD3 mAb (PerCp). For Kv1.3 detection, ShK-F6CA (0.3 µg/ml; Bachem AG, Bubendorf, Switzerland) was incubated together with mAb against cell surface proteins for 30 min at room temperature .

Stimulation of OT-II CD4 + T Cells In Vitro
Lymph node cells and red blood cell-lysed splenocytes from OT-II mice were pooled and 2 × 10 5 cells seeded per well of a 96-well round bottom plate (Greiner). OVA 323-339 peptide (OVAp, Charité Berlin) was added at 1 or 0.1 µM. For each condition six technical repeats were set up. On day three, culture supernatant was harvested and frozen at -70 • C. Cells were stained for CD4 and CD25 expression and absolute cell numbers were determined by FACS using counting beads. To generate Th1 cells pooled spleen/lymph node cells from an OT-II mouse were depleted of CD4 + CD25 + regulatory T cells using anti-CD25 biotin (5 µg/ml, BD) and Streptavidin-beads and passage over an LD column (both Miltenyi). CD25-depleted spleen/lymph node cells were then seeded at 1 × 10 6 cells/well (48-well plate, Greiner, final volume: 1 ml, 3 × 10 6 cells in total), OVAp was added at 1 µM, recombinant mouse IL-12 (R&D Systems, Minneapolis, MN, USA) at 10 ng/ml and goatanti-mouse IL-4 (R&D Systems) at 10 µg/ml. To obtain 'Th0' cells the CD25-depleted spleen/lymph node cells were stimulated with OVAp only. After five days of culture, CD4 + T cells were magnetically purified (CD4 + Isolation kit, Affymetrix, according to manufacturer's instructions) and cultured for another two days in the presence 0.1 µM Proleukin R (Novartis) (48-well plate, 5 × 10 5 cells/well). Afterward, the cells were harvested and cocultured with T cell-depleted splenocytes (anti-CD90.2 beads, LD column, Miltenyi) isolated from a WT C57BL/6 mouse (96well round-bottom plate; 1 x 10 5 T cell-depleted splenocytes per well, 1 × 10 4 Th1 cells per well, triplicates). OVAp and Pra1 were added to these cultures in different concentrations. Every day 15 µl of culture supernatant were harvested per well to determine cytokine concentrations. On day three the Th1 cells were restimulated with phorbol myristate acetate (5 ng/ml) and ionomycin (500 ng/ml) for two hours at 37 • C/5% CO 2 (v/v) before addition of Brefeldin A (10 µg/ml; all Sigma) and incubation for another two hours followed by cell surface staining for CD4, fixation, and permeabiliziation of the cells (Fix/Perm and Perm buffers, ebioscience) and intracellular staining for IFNγ expression (anti-IFNγ Alexa Fluor 488, clone XMG1.2, Biolegend).

Cytokine Detection in Culture Supernatants
Concentrations of the indicated cytokines were determined in culture supernatants using LEGENGplex TM (Biolegend) according to the manufacturer's instructions.

Statistics
Summary graphs were generated and statistical testing was done using Excel © 14.4.1 (Microsoft) and Prism 4.0c © (GraphPad). P < 0.05 was considered statistically significant.

Ethics Statement
Stadt Würzburg (City of Würzburg) and UK Home Office (PPL 30/3038) approved breeding of the mice used in this study and the animals were culled by Annex IV approved techniques in accordance with Directive 2010/63/EU.

Pra1 Directly Binds to Mouse CD4 + T Cells in a Zinc-Dependent Manner
As Pra1 expression of C. albicans is induced upon contact with human cells and as it has already been shown to strongly modulate innate immunity (Zipfel et al., 2011), we studied direct binding of Pra1 to mouse CD4 + T cells in vitro. We used recombinantly expressed Pra1 purified from Pichia pastoris for staining and found that Pra1 bound to all splenocytes in a dose-dependent manner ( Figure 1A, left histogram). Among total splenocytes CD11b + CD3 − monocytic cells bound Pra1 particularly well ( Figure 1A, middle left histogram), which was expected as complement receptor 3 (CR3, Mac1, CD11b/CD18) had been identified as a cellular receptor for Pra1 on mouse leukocytes (Soloviev et al., 2007(Soloviev et al., , 2011. Splenic B (Figure 1A, middle right histogram) and T cells (Figure 1A, right), i.e., CD4 + and CD8 + T cells (Figure 1B), also clearly bound Pra1, albeit to a lesser extent than the monocytic cells.
As Pra1 binds zinc (Citiulo et al., 2012) we tested whether zinc influences Pra1 binding to mouse CD4 + T cells. Zn 2+ which is found in serum at a concentration of 10 µM (Feske et al., 2012) and beyond increased Pra1 binding to mouse CD4 + T cells (Figures 2A-C) with plateau levels of binding reached after 30 min of incubation ( Figure 2D). Moreover, a pra1 deletion mutant encoding a Pra1 protein lacking the putative zinc-binding domain (Pra1 238-299) (Citiulo et al., 2012) showed almost no binding to mouse CD4 + T cells ( Figure 2E).
The zinc binding capacity of Pra1 is shared by its homolog in A. fumigatus, i.e., the Aspf2 protein (Citiulo et al., 2012). We, therefore, used recombinantly expressed (P. pastoris) and purified Aspf2 and tested whether Aspf2 also directly binds to mouse CD4 + T cells. Aspf2, in contrast to Pra1, however, did not bind to the mouse T cells even when ZnCl 2 was added to the buffer (Figure 3). Thus, Pra1, but not Aspf2, directly binds to mouse CD4 + T cells and Pra1 binding is enhanced in the presence of extracellular zinc.

Complement Regulatory Proteins Expressed by Mouse CD4 + T Cells Do Not Interact with Pra1
So far, only complement receptor 3 (CR3, Mac1, CD11b/CD18) has been identified as a cellular receptor for Pra1 on mouse leukocytes (Soloviev et al., 2007(Soloviev et al., , 2011. As the staining pattern of Pra1 showed that Pra1 binds similarly well to all mouse CD4 + T cells ( Figure 1B) we hypothesized that a complement regulatory protein expressed by all mouse T cells might be the receptor for Pra1. Therefore, we analyzed Pra1 binding to CD4 + T cells of CD55 −/− mice in more detail as CD55, Crry, and CD59a are the three complement-regulatory proteins expressed by mouse T cells (Miwa and Song, 2001). While CD4 + T cells of CD55 −/− mice were clearly devoid of CD55 expression at the cell surface ( Figure 4A) binding of Pra1 was not reduced in the absence of CD55 (Figure 4B). Moreover, addition of zinc also increased binding of Pra1 to mouse CD4 + T cells of CD55 −/− mice ( Figure 4B). Apart from CD55 −/− mice we also studied binding of Pra1 to CD4 + T cells of Crry −/− and CD59a −/− mice, which was also not reduced ( Figure 4C). Therefore, Pra1 does not seem to interact with any of the three complement regulatory proteins expressed on the surface of mouse CD4 + T cells.

Pra1 Binding Co-stimulates Mouse CD4 + T Cells
To gain further insight into the functional consequences of Pra1 binding to CD4 + T cells, we first studied its impact on T cell activation and proliferation in vitro. To avoid confounding effects FIGURE 2 | Binding of C. albicans Pra1 to mouse CD4 + T cells is enhanced by extracellular zinc. (A) Representative histograms depicting Pra1 binding to gated mouse CD4 + T cells (total lymph node cells) in the presence of different concentrations of ZnCl 2 (black: Pra1; gray: background staining control). (B) Summary graph depicting Pra1 (black) versus background staining (gray) of CD4 + T cells in the presence of different concentrations of ZnCl 2 (n = 6, two-sided t-test, * p < 0.05). (C) Summary graph showing enhanced binding of Pra1 to mouse CD4 + T cells in the presence of ZnCl 2 (relative MFI(Pra1) = MFI(Pra1)/MFI(background); means ± SD; n = 6; two-sided t-test, * p < 0.05). (D) Pra1 on-kinetics at 37 • C. Lymph node cells were incubated with Pra1 for the indicated periods of time before we detected binding of Pra1 to CD4 + T cells (means ± SD of n = 3 separate experiments; two-sided t-test). (E) Comparison of Pra1wt (white columns) and a mutated Pra1 lacking the zinc-binding domain (aa 238-299, black columns) binding to gated CD4 + T cells in the presence of 10 µM ZnCl 2 (left) or 0 and 100 µM ZnCl 2 (right). Means + SD (n = 3-6 individual measurements). Two-sided t-test. * p < 0.05, * * p < 0.01, * * * p < 0.001. through the interaction of Pra1 with CD11b/CD18 expressed by monocytic cells in our cultures, we FACS-sorted mouse CD4 + T cells which lack CD11b/CD18 to more than 99% purity. Stimulation of these highly pure CD4 + T cells by plate-bound anti-CD3 mAb and titrated amounts of Pra1 led to a dosedependent increase in proliferation and CD25 expression similar to what we observed by adding an anti-CD28 mAb (Dennehy et al., 2006) (Figures 5A,B). Moreover, Pra1 truly induced a co-stimulatory signal in the T cells as in the absence of CD3 stimulation Pra1 did not activate the cells (Figure 5C). The same effect was observed for the anti-CD28 mAb ( Figure 5C). Binding of Pra1 to mouse CD4 + T cells, thus, enhanced T cell activation and proliferation, which comprise the first steps of the adaptive immune response.

Cytokine Secretion by In Vivo Generated Mouse CD4 + Memory T Cells is Inhibited in the Presence of Pra1
While the activation of naïve T cells and clonal expansion mark the beginning of the CD4 + T cell response, secretion of cytokines such as IFNγ characterize its effector and FIGURE 3 | The Pra1 homolog of A. fumigatus, Aspf2, does not bind to mouse CD4 + T cells. Representative staining of Aspf2 on gated CD4 + T cells derived from the spleen as performed before with Pra1. Aspf2-and Pra1-staining on cells from the same mouse with Pra1-and Aspf2-(black) and background staining (gray). The bar diagram shows relative MFI of the Aspf2-signal with and without ZnCl 2 (relative MFI(Aspf2) = MFI(Aspf2)/MFI(background); means ± SD; n = 3). memory phase. We, therefore, analyzed cytokines in the supernatants of purified CD4 + T cells, containing in vivo generated memory T cells, stimulated via plate-bound anti-CD3 mAb and soluble Pra1 or anti-CD28 mAb (Dennehy et al., 2006) (Figure 6A). In contrast to its co-stimulatory effect on T cell activation and proliferation Pra1 suppressed secretion of both Th1 and Th2 cytokines ( Figure 6A). Only IL-17 secretion appeared not to be affected, while secretion of IL-10 was below the detection limit in these experiments. Seemingly at odds with our observation concerning expression of the IL-2 receptor α-chain, CD25 (Figure 5), IL-2 concentrations were also reduced in the presence of Pra1. We assume that this reflects increased IL-2 consumption through increased receptor expression rather than reduced IL-2 production (Malek, 2008) uniting these two findings. To further test the capacity of Pra1 to inhibit cytokine secretion we added Pra1 to purified CD4 + T cells which we co-stimulated with anti-CD3/anti-CD28 mAb-coated Dynabeads R (Figure 6B). Even under these conditions, which more faithfully mimic T cell-antigen presenting cell interactions than stimulation via plate-bound antibodies, Pra1 reduced cytokine, i.e., IFNγ, secretion by the CD4 + T cells (Figure 6B). The same was true for the supernatant of cultured C. albicans containing the whole array of secreted fungal proteins ( Figure 6B).
Apart from binding to CD4 + T cells, Pra1 interacts with CD11b/CD18 integrin (Mac1) expressed by monocytic and granulocytic cells (Soloviev et al., 2011). To test whether Pra1 also suppresses IFNγ secretion in the presence of Mac1-expressing antigen-presenting cells (APCs) we stimulated total splenocytes from T cell receptor-transgenic OT-II mice with 1 µM OVApeptide 323-339 in the presence of 100 or 1 ng/ml Pra1 (Figure 6C). Also under these conditions Pra1 inhibited IFNγ secretion by the OT-II CD4 + T cells.
Both in the presence of recombinant Pra1 as well as C. albicans supernatant, secretion of cytokines by mouse CD4 + T cells was, thus, reduced.
Depending on the Strength of the TCR Signal Pra1 Also Reduces Secretion of IFNγ by In Vitro Generated Th1 Cells T cells isolated from healthy mice producing effector cytokines are by definition mostly resting memory T cells. During acute invasive C. albicans infection or C. albicans-induced inflammation the fungus, however, mainly encounters effector T cells. Therefore, we first deliberately generated OT-II Th1 effector cells during a five-day culture in vitro followed by a two-day resting phase and subsequent re-stimulation of the Th1 cells in the presence of APCs and different concentrations of peptide antigen and Pra1 (Figure 7A). Addition of Pra1 to Th1 cells stimulated with 0.1 µM OVA peptide reduced IFNγ secretion into the supernatant (Figure 7A, middle), while this was not the case at 1 µM OVA peptide (Figure 7A, right). Analyzing intracellular IFNγ expression by the Th1 cells after PMA/ionomycin re-stimulation, further, showed that the reduced secretion of IFNγ into the culture supernatant in the presence of Pra1 was not due to a per se lower capacity of the Th1 cells to produce IFNγ. Without OVAp re-stimulation the expression of IFNγ by the Th1 cells was, however, reduced suggesting that Pra1 increases the threshold for stimulationinduced cytokine secretion by CD4 + T cells. Incubation of Th1 cells with Pra1 showed, in comparison to OT-II CD4 + T cells cultured under Th0 conditions in parallel, that Th1 cells bind Pra1 better than Th0 cells (Figures 7C,D) suggesting that differentiated effector memory Th1 cells are a primary target of Pra1. In autoreactive pathogenic T cells Kv1.3 has been shown to be the main voltage-gated potassium channel and blocking the channel with the ShK peptide inhibits the autoreactive pathogenic T cells in animal models of autoimmunity in vivo (Beeton et al., 2001) and in cell cultures of human T cells in vitro . Using a fluorescently labeled ShK peptide we observed that the Th1 cells expressed more Kv1.3 channels than Th0 cells and that Pra1 binding and Kv1.3 expression were positively correlated in Th1 cells ( Figure 7E). Pra1, thus, preferentially bound to effector/memory Th1 cells inhibiting IFNγ secretion provided TCR stimulation did not surpass a certain threshold.

DISCUSSION
In this study, we describe the direct interaction of the secreted C. albicans protein Pra1 with mouse CD4 + T cells. Binding of Pra1 to the CD4 + T cells was enhanced by extracellular Zn 2+ . Moreover, Pra1 binding inhibited cytokine secretion from CD4 + T cells in vitro thus constituting a novel immune evasion mechanism for C. albicans.
In line with its known capacity to scavenge Zn 2+ ions (Citiulo et al., 2012) Pra1 bound more efficiently to mouse CD4 + T cells in the presence of extracellular zinc than in its absence (Figure 1). This activity was in contrast to what we observed for Aspf2, the zinc-binding Pra1-homolog of A. fumigatus (Citiulo et al., 2012). Aspf2 did not bind to mouse CD4 + T cells -either in the presence or absence of Zn 2+ (Figure 3). As both proteins carry a HIStag, which, of course, by itself is capable of binding Zn 2+ (Evers et al., 2008), the enhanced binding of Pra1 to mouse CD4 + T cells after addition of ZnCl 2 was not merely mediated by the HIS-tag. Moreover, even under conditions where we did not add ZnCl 2 during the staining procedure we detected a positive signal for Pra1 binding (Figures 1, 2). This data implies that the Pra1 binding to the surface of the CD4 + T cells is not strictly zincdependent and/or that free zinc present in preparations of lymph node cells and splenocytes might be sufficient to allow for Pra1 binding.
The molecular basis for the enhanced Pra1 binding mediated by ZnCl 2 is so far not clear. We envisage that Zn 2+ binding might induce a conformational change in Pra1 as has been described for many other Zn 2+ -binding proteins (Ebert and Altman, 2008). Such a structural change has, however, not yet been described for Pra1.
While the receptor for Pra1 on the surface of mouse CD4 + T cells is still elusive, CR3 (CD11b/CD18, Mac-1) expressed by neutrophils and monocytic cells has been shown to bind Pra1 FIGURE 5 | Pra1 co-stimulates mouse CD4 + T cells. (A) CFSE dilution and CD25 expression by mouse CD4 + T cells after a 3 day culture in the presence or absence of plate-bound αCD3 mAb and Pra1 (1 ng/ml) or αCD28 mAb (1 µg/ml) added in solution. (B) Summary graph depicting a dose-dependent increase in co-stimulation in the presence of plate-bound αCD3 mAb and Pra1 (0% = frequency CFSE low CD25 + with αCD3 mAb only; 100% = frequency CFSE low CD25 + with αCD3 + 10 µg/ml αCD28 mAb). (C) Direct stimulatory activity of Pra1 in the absence of plate-bound αCD3 (0% = frequency CFSE low CD25 + medium only; 100% = frequency CFSE low CD25 + with plate-bound αCD3 mAb). (B,C): Means ± SD (n = 3 individual experiments). Two-sided t-test: * p < 0.05. and that this binding is important to protect mice after systemic C. albicans infection (Soloviev et al., 2011). On mouse CD4 + T cells it is, however, not a complement regulatory protein that interacts with Pra1 (Figure 4). Therefore, it is unlikely that modulation of complement activation, which has been shown to crucially contribute to T cell stimulation and differentiation (Arbore and Kemper, 2016), accounts for the effects of Pra1 on mouse CD4 + T cells. Analysis of Kv1.3 expression in parallel to Pra1 binding to in vitro polarized CD4 + Th1 cells, however, showed that cells with the highest capacity to bind Pra1 also expressed high levels of Kv1.3 (Figure 7). While we do not, yet, know whether Kv1.3 is a receptor for Pra1 it may not be the only molecule Pra1 interacts with on the T cell surface. Kv1.3 expression cannot be detected on resting T cells by FACS using the ShK-F6CA peptide , while Pra1 binding to resting T cells is detectable by flow cytometry as detailed in this study. Functionally, Pra1 might interfere with K + currents through Kv1.3 by direct binding to the channel or by binding in the vicinity of Kv1.3 and 'delivering' Zn 2+ ions. Extracellular Zn 2+ binds to Kv1.3 inhibiting the transport of K + ions through the channel Mozrzymas, 2002, 2006).
Apart from directly interacting with CD4 + T cells, Pra1 could also modulate T cell responses by binding to APCs via interaction with CD11b/CD18 (Soloviev et al., 2011) or via the still unknown Pra1 receptor also expressed on T cells. Therefore, it was important to study the effects on cytokine secretion by CD4 + T cells in the presence of APCs. Irrespective of whether APCs were present in our assays Pra1 inhibited cytokine secretion FIGURE 6 | Modulation of cytokine secretion by Pra1 upon polyclonal and antigen-specific CD4 + T cell stimulation. (A) The left panel shows the absolute amount of cytokines secreted by purified WT CD4 + T cells after three days of stimulation with plated-bound anti-CD3-mAb (2.5 µg/ml) either alone or together with Pra1 or anti-CD28 mAb (clone E18, 10 µg/ml) added in solution. The right column shows the amounts of secreted cytokines normalized to the 'anti-CD3 mAb only' cultures (=100%; first bar in the left panel). Means + SD of n = 5 individual experiments are shown. (B) IFNγ secretion upon stimulation of purified CD4 + T cells with anti-CD3/anti-CD28 mAb-coated Dynabeads R . Pra1 was added at 1 ng/ml and the C. albicans supernatant was diluted 1:25. Means + SD of triplicate cultures are shown. The experiment was repeated with similar result. (C) Lymph node cells from OT-II mice were stimulated with 1 µM OVAp in the absence or presence of Pra1 as indicated before supernatants were harvested on day three and IFNγ concentrations determined (means ± SD of triplicate cultures; experiment was repeated with similar result). (A-C) Two-sided t-test. * p < 0.05, * * p < 0.01, * * * p < 0.001. by the CD4 + T cells (Figures 6, 7) suggesting that the direct interaction of Pra1 with the CD4 + T cells was also the crucial event in the cultures containing APCs.
Recombinant Pra1 and supernatant of C. albicans cultures inhibited IFNγ release from CD4 + T cells (Figure 6). While we do not know to what extent Pra1 contributes to the overall inhibitory effect of the C. albicans supernatant this observation highlights that C. albicans, through its secretome, modulates CD4 + T cell responses. Further experimentation is required to delineate whether Pra1 is the only C. albicans protein mediating these effects or, more likely, whether other secreted fungal proteins also contribute to effector T cell inhibition.
Apart from Th1 cells, Th17 cells also crucially contribute to anti-fungal immunity either through direct effects or by FIGURE 7 | IFNγ secretion by OT-II Th1 cells in the presence of Pra1. (A) In vitro generated OT-II Th1 cells were re-stimulated in the presence of the indicated amounts of OVAp, Pra1 and APCs. Culture supernatants were harvested on days one to three and IFNγ content determined. Means ± SD of triplicate cultures are shown. The experiment was repeated with similar result. An ANOVA was used to compare cultures with to those without Pra1. (B) At the end of the re-stimulation cultures, we determined IFNγ expression by OT-II Th1 cells intracellularly. Means ± SD of three independent experiments are shown. A two-sided t-test was used to compare groups. (C) Representative Pra1 staining (black; gray: control without Pra1) of OT-II CD4 + T cells stimulated with OVAp under Th0 (left; 1% IFNγ + ) or Th1 conditions for 5 days (right, 53% IFNγ + ). (E) Detection of Kv1.3 expression via staining in parallel to Pra1 binding in the samples shown in (C,D). (D,E): Means ± SD of triplicate analyses. The experiment was repeated twice with similar result. A two-sided t-test was used to compare groups. * p < 0.05, * * p < 0.01, * * * p < 0.001.
supporting Th1 versus Th2 cell differentiation (Romani, 2011;Zelante et al., 2016). In contrast to other cytokines, IL-17 release from CD4 + T cells was not reduced in the presence of Pra1 (Figure 6). This might have to do with the degree of TCR signal strength required to induce optimal cytokine release from different CD4 + T helper cell subpopulations. For Th1 cells we observed that strong TCR stimulation overcame Pra1-induced suppression of cytokine release (Figure 7). As maximal IL-17 release, in contrast to IFNγ release, has been reported to require low TCR stimulation (Purvis et al., 2010) further experimentation is required to determine whether, indeed, Pra1 differentially regulates cytokine release from Th1 and Th17 cells.
In summary, our data identify Pra1 as an inhibitor of mouse CD4 + effector T cell function in vitro, thus, mediating evasion of C. albicans from potentially harmful CD4 + T cell responses. While subversion of the CD4 + T cell response during commensalism might be of mutual benefit for C. albicans and the host, during invasive infection/sepsis blocking protective CD4 + T cell immunity might worsen clinical outcome. Therefore, the findings of our study suggest that therapeutic targeting of soluble Pra1 might enhance CD4 + T cell responses protecting the host from invasive C. albicans infections.

AUTHOR CONTRIBUTIONS
AB designed research studies, conducted experiments, acquired and analyzed data, and wrote the paper. PD provided reagents, designed research studies, and interpreted data. SW conducted experiments, acquired and analyzed data. TRH provided reagents, designed research studies, and interpreted data. WS provided reagents and interpreted data. PH provided reagents and designed research studies. AB provided reagents, designed research studies, and interpreted data. TH designed research studies and analyzed and interpreted data. PZ provided reagents, designed research studies, interpreted data, and wrote the manuscript. NB designed research studies, analyzed, and interpreted data and wrote the paper.

FUNDING
This study was funded by a grant from the DFG (CRC124 FungiNet -project C6 and A1). The publication of the study was funded by the DFG and the University of Würzburg in the funding programme Open Access Publishing.