Heterogeneity of Multifunctional IL-17A Producing S. Typhi-Specific CD8+ T Cells in Volunteers following Ty21a Typhoid Immunization

Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, continues to cause significant morbidity and mortality world-wide. CD8+ T cells are an important component of the cell mediated immune (CMI) response against S. Typhi. Recently, interleukin (IL)-17A has been shown to contribute to mucosal immunity and protection against intracellular pathogens. To investigate multifunctional IL-17A responses against S. Typhi antigens in T memory subsets, we developed multiparametric flow cytometry methods to detect up to 6 cytokines/chemokines (IL-10, IL-17A, IL-2, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-1β (MIP-1β)) simultaneously. Five volunteers were immunized with a 4 dose regimen of live-attenuated S. Typhi vaccine (Ty21a), peripheral blood mononuclear cells (PBMC) were isolated before and at 11 time points after immunization, and CMI responses were evaluated. Of the 5 immunized volunteers studied, 3 produced detectable CD8+ T cell responses following stimulation with S. Typhi-infected autologous B lymphoblastoid cell lines (B-LCL). Additionally, 2 volunteers had detectable levels of intracellular cytokines in response to stimulation with S. Typhi-infected HLA-E restricted cells. Although the kinetics of the responses differed among volunteers, all of the responses were bi- or tri-phasic and included multifunctional CD8+ T cells. Virtually all of the IL-17A detected was derived from multifunctional CD8+ T cells. The presence of these multifunctional IL-17A+ CD8+ T cells was confirmed using an unsupervised analysis program, flow cytometry clustering without K (FLOCK). This is the first report of IL-17A production in response to S. Typhi in humans, indicating the presence of a Tc17 response which may be important in protection. The presence of IL-17A in multifunctional cells co-producing Tc1 cytokines (IL-2, IFN-γ and TNF-α) may also indicate that the distinction between Tc17 and Tc1 responses in humans is not as clearly delineated as suggested by in vitro experiments and animal models.


Introduction
Salmonella enterica serovar Typhi (S. Typhi), a human restricted pathogen, is the causative agent of typhoid fever. S. Typhi is a Gram-negative, facultative intracellular pathogen that poses a significant threat to public health, particularly in the developing world [1,2]. Typhoid fever is responsible for an estimated 21 million illnesses and 200,000 deaths annually and increasing antibiotic resistance among S. Typhi isolates has further exacerbated the problem [1,2,3,4,5,6,7,8]. T cells, and particularly CD8+ T cells are thought to play an important role in the immune response against S. Typhi by producing interferon-c (IFN-c) and other T helper (Th) 1/T cytotoxic (Tc) 1 cytokines [9,10,11,12,13,14,15,16,17], as well as killing S. Typhi infected cells [10,18]. Additionally, multifunctional CD8+ T cells have been identified in volunteers immunized with live-attenuated typhoid vaccine Ty21a [13]. Of note, the latter study has shown multiphasic cytokine production by CD8+ T cells in response to human leukocyte antigen E (HLA-E)-restricted antigenic stimulation [13].
Therefore, to evaluate the hypothesis that IL-17A may play a role in protection from S. Typhi infection, we investigated the presence of IL-17A producing CD8+ T cells in peripheral blood mononuclear cells (PBMC) obtained from Ty21a recipients following ex vivo stimulation with S. Typhi-infected autologous Blymphoblastoid cell lines (B-LCL) or an S. Typhi-infected HLA-E restricted cell line. Furthermore, given the prominent role that multifunctional T cells have been shown to have in the host defense from other human infections [29,30,31], we investigated the concomitant production of IL-17A with 5 other cytokines/ chemokines (IL-10, IL-2, IFN-c, tumor necrosis factor-a (TNF-a), and macrophage inflammatory protein-1b (MIP-1b) as a first step to investigate the potential role of multifunctional T cells in protection from S. Typhi infection.

Volunteers and Isolation of Peripheral Blood Mononuclear Cells (PBMC)
PBMC collected from 6 healthy adult volunteers, recruited from the Baltimore-Washington area and University of Maryland, Baltimore campus, were used in this study. Written informed consent was obtained and all procedures were approved by the University of Maryland, Baltimore IRB. PBMC were isolated immediately after blood draws by density gradient centrifugation and cryopreserved in liquid nitrogen following standard techniques [18,32].

Target/stimulator Cells
Epstein Barr virus (EBV)-transformed B-LCL were generated from PBMC obtained from Ty21a vaccinees and control volunteers as previously described [18]. Briefly, B-LCL were established using supernatants from the B95.8 cell line (ATCC CRL1612; American Type Culture Collection) as the source of EBV. PBMC from each volunteer were incubated with EBV containing supernatant and cyclosporine (0.5 mg/mL; Sigma, Saint Louis, MO) at 37uC with 5% CO 2 for 2-3 weeks. B-LCL were maintained in culture or cryopreserved until use.

Infection of Target/stimulator Cells
Target cells were infected by incubation with wild-type S. Typhi strain ISP1820 in RPMI without antibiotics for 3 hours at 37uC with 5% CO 2 as previously described [18]. On the day following infection the cells were gamma irradiated (6000 rad). To confirm that targets were infected with S. Typhi, cells were stained with anti-Salmonella common structural Ag (CSA-1)-FITC (Kierkegaard & Perry, Gaithersburg, MD) and analyzed by flow cytometry using an LSR-II instrument (BD). The percentage of cells infected with S. Typhi was recorded for each experiment. Infected targets were only used if infection was .30% of viable cells.

Ex vivo Stimulation
PBMC were thawed and rested overnight at 37 o C, 5% CO 2 in complete media (RPMI 1640 media (Gibco) supplemented with 100 U/mL penicillin (Sigma), 100 mg/mL streptomycin (Sigma), 50 mg/mL gentamicin (Gibco), 2 mM L-glutamine (Gibco), 2.5 mM sodium pyruvate (Gibco), 10 mM HEPES buffer (Gibco), and 10% fetal bovine serum (Gemini Bioproducts,)) at a concentration of 1610 6 cells/mL in 6 or 12 well plates. Cells were then resuspended in complete media and stimulated with S. Typhi-infected autologous B-LCL or S. Typhi-infected HLA-E restricted AEH cells. Media alone and uninfected autologous B-LCL or HLA-E restricted AEH cells were used as negative controls. Staphylococcal enterotoxin B (SEB) (10 mg/mL; Sigma) was used as a positive control. Cells were incubated at 37uC in 5% CO 2 . After 2 hours, Golgi Stop (containing monensin) and Golgi Plug (containing brefeldin A) from BD were added at concentrations of 0.5 ml/mL and cultures continued overnight at 37uC in 5% CO 2 .

Statistical Analyses
All tests were performed using GraphPad Prism version 5.03 (Graphpad Software). Comparisons between groups were performed using Mann-Whitney and Kruskal-Wallis tests. In flow cytometry experiments a response was considered significant if the differential in the number of positive events between experimental (S. Typhi-infected) and negative control (non-infected) cultures was significantly increased by Chi-square tests. P values ,0.05 were considered significant.

Kinetics of S. Typhi-specific CD8+ T Cells in Response to Stimulation with S. Typhi-infected Autologous Cells
Previous studies have indicated that CD8+ T cells are a major component of the CMI response to S. Typhi [10,14,15,18]. Therefore we set out to determine the kinetics of CD8+ T cell responses to in vitro stimulation with autologous S. Typhi-infected cells following Ty21a immunization. Five healthy adult volunteers received 4 doses of the licensed oral, live-attenuated S. Typhi vaccine Ty21a on days 0, 2, 4, and 7. One volunteer did not receive Ty21a (negative control). PBMC were isolated before immunization and at 11 time-points up to 1 year after immunization (days 2, 4, 7, 10, 14, 28, 42, 56, 120, 180, and 360). Following isolation, PBMC were stimulated with S. Typhiinfected autologous B-LCL. Uninfected B-LCL and SEB were used as negative and positive controls respectively. PBMC were stained with monoclonal antibodies against CD3, CD4, CD8, CD14, CD19, CD45RA, and CD62L followed by intracellular staining for CD69, IL-10, IL-17A, IL-2, IFN-c, TNF-a, and MIP-1b. Due to unavailability of the antibody, MIP-1b staining was not performed in 2 of the volunteers (48 s and 50 s). The samples were then analyzed by multichromatic flow cytometry. The gating strategy is shown in Figure S1. CD3+ CD8+ T cells were categorized by their expression of CD62L and CD45RA into naïve T cells (T N ; CD62L+ CD45RA+), T central memory cells (T CM ; CD62L+ CD45RA-), T effector memory (T EM ; CD62L-CD45RA-), and T effector memory CD45RA+ (T EMRA ; CD62L-CD45RA+) as previously described ( Figure S1) [34]. We observed the intracellular production of multiple cytokines to be predominantly by the T EM subset with a smaller contribution by the T EMRA subset ( Figure 1).
Low levels of cytokine production were detected in PBMC stimulated with uninfected autologous B-LCL and this background was subtracted to calculate the S. Typhi-specific response. There was some variable baseline responsiveness on day 0 in most volunteers, likely due to cross-reactivity with other enteric Gram negative rods. Therefore, the net value (day x post immunizationday 0) was used to normalize the data. We found that 3 of the 5 immunized volunteers (60%) showed detectable intracellular cytokine responses above baseline responsiveness at day 0 ( Figure 2 and Figure S2). This is consistent with our previous data using PBMC from subjects immunized with Ty21a and, interestingly, with most field studies which report a vaccine efficacy of 65-70% [10,13,35]. Intracellular IL-10 was not detected in any of the volunteers at any time-point. MIP-1b was measured in only 2 of the 3 responders (53 s and 54 s). There was significant production of MIP-1b by both T EM and T EMRA from volunteer 53 s (Figure 2 A & B); however, volunteer 54 s produced no detectable MIP-1b ( Figure 2 C & D). CD8+ T EM from all 3 volunteers (53 s, 54 s, and 50 s) produced high levels of TNF-a and IFN-c (5-15% of circulating T EM at the peak times), followed by IL-2 (4-8% of circulating T EM at the peak times), and then IL-17A (2-5% of circulating T EM at the peak times) ( Figure 2). Intracellular IL-17A production was found in all 3 responders with kinetics that followed those of the other cytokines/chemokines measured (except MIP-1b) ( Figure 2). The kinetics of the responses varied considerably among volunteers; however, all 3 volunteers showed bi-or tri-phasic responses. Early peaks were observed in volunteers 53 s and 50 s at days 4 to 7 post immunization (

Differences in Kinetics of S. Typhi-specific HLA-E Restricted CD8+ T Cells
Here we investigated whether IL-17A was produced following stimulation by HLA-E restricted CD8+ T cells, as well as defined the kinetics of this response. As described for PBMC stimulated with S. Typhi-infected autologous B-LCL, PBMC from immunized volunteers were stimulated with the S. Typhi-infected HLA-E restricted cell line (721.221.AEH), stained, and analyzed by multichromatic flow cytometry. The same gating strategy was applied ( Figure S1). We found that T EM and to a lesser extent T EMRA subsets were the major sources of intracellular cytokine production ( Figure 3). In contrast to stimulation with S. Typhiinfected autologous B-LCL, HLA-E restricted stimulation resulted in responses in 2 of the volunteers, 53 s and 54 s (Figure 4 and Figure S3). This is unlikely to be the result of technical difficulties.  (Figure 4 C). TNF-a (5.29-6.37%) and IFN-c (5.31-7.32%) had the highest percentage of cells with detectable intracellular cytokines, followed by IL-2 (2.87-3.85%) and IL-17A (2.64-4.71%) at peak time points (Figure 4). Although the kinetics were also bi-or tri-phasic, there were differences between the peak timepoints following stimulation with S. Typhi-infected autologous B-LCL and HLA-E restricted cells (Figures 2 & 4). Volunteer 54 s showed a bi-phasic response to stimulation with S. Typhi-infected autologous B-LCL (

S. Typhi-specific CD8+ T Cells are Primarily Multifunctional
Because recent reports suggested that multifunctional T cells are likely to play a prominent role in protection from infection [25,26,27], we next investigated the multifunctionality of CD8+ T cell responses to Ty21a. Using the FCOM function of WinList (Verity software) we analyzed all possible combinations (64 total) of the 6 cytokines/chemokines measured (IL-10, IL-17A, IL-2, IFNc, TNF-a, and MIP-1b) at each time-point. Only combinations with at least 1 time-point $0.5% of CD8+ T EM or T EMRA cytokine positive cells are shown. This percentage represents a minimum of ,10-50 events for T EMRA and T EM respectively. Results from a representative volunteer (53 s) are shown in response to stimulation with S. Typhi-infected autologous cells ( Figure 6) and S. Typhi-infected HLA-E restricted cells (Figure 7). Data from the other responding volunteers are shown in Figures  S3,S4,S5. Due to the complexity of the data (64 combinations for each of 12 time-points) we determined the median value for each combination that was $0.5% based on all time-points ( Figures 6A,  7A, S3A, S4A, S5A). Each combination is shown on the X-axis with the values from each time-point (in % positive cells) on the Yaxis. The median value was determined for each combination and the kinetics of the top 5 combinations are shown (Figures 6B, 7B). Multifunctional responses predominated over single cytokine producing cells in all volunteers (Figures 6 and 7, and S3,S4,S5). As demonstrated in Figure 6, 4 of the top 5 combinations identified were multifunctional for volunteer 53 s in response to autologous stimulation. All of the top 5 combinations for volunteer 53 s produced MIP-1b (either singly or in combination with other cytokines). The top 5 combinations for volunteer 53 s were the same for both autologous and HLA-E restricted stimulation (Figures 6 & 7). In contrast, in volunteer 54 s the top combinations differed depending on the type of stimulation (Figures S3 and S4). Volunteer 50 s did not respond to HLA-E restricted stimulation.
IL-17A Producing Cells are Multifunctional and coproduce IL-2, IFN-c, TNF-a, and/or MIP-1b As shown above, IL-17A production was detected in all of the responding volunteers following stimulation with both autologous and HLA-E restricted cells (Figures 2, 4, and S3,S4,S5). To further characterize the IL-17A producing cells, we studied their multifunctional characteristics in further detail. We found that virtually all of the IL-17A producing cells were multifunctional, coproducing IL-2, IFN-c, TNF-a, and/or MIP-1b (

Unsupervised Analysis Using Flow Cytometry Clustering without K (FLOCK) Confirms the Multifunctionality of S. Typhi-specific CD8+ IL-17A+ T Cells
To further examine the multifunctional IL-17A+ populations identified by conventional, user-guided, flow cytometry analyses, we used the novel unsupervised analysis program FLOCK. FLOCK uses computational methods to determine the number of unique populations in multidimensional flow cytometry data [36]. A subset of time-points (days 0, 7, 10, and 42 post immunization) for volunteers 53 s and 54 s following stimulation Figure 3. Detection of intracellular cytokines/chemokines produced by CD8+ T cell memory subsets in response to stimulation with S. Typhi-infected HLA-E restricted cells. Histograms from a representative volunteer (53 s at day 7 post infection) showing the production of 6 cytokines/chemokines by memory T cell subsets following stimulation with S. Typhi-infected AEH cells. CD69 (marker of recent activation) is displayed on the y-axis and each of the 6 cytokines/chemokines measured are displayed on the x-axes. Column 1 represents total CD8+ T cells, column 2 represents T CM , column 3 represents T N , column 4 represents T EM , and column 5 represents T EMRA . The percentage of positive cells is shown for the indicated region in each cytogram. doi:10.1371/journal.pone.0038408.g003 with S. Typhi-infected B-LCL were analyzed. The same data that were analyzed for intracellular detection of IL-10, IL-17A, IL-2, IFN-c, TNF-a, and MIP-1b by conventional, user-guided methods (Figures 1, 6, and S3) were analyzed by FLOCK. Prior to FLOCK analyses, gating was performed as described in Figure  S1 to select CD3+ CD8+ T EM events. Data for the 4 selected timepoints for each volunteer were uploaded to the ImmPort website (http://immport.niaid.nih.gov) and FLOCK analyses performed. The number of unique populations varied at different time-points and between volunteers (9-29 individual populations). In order to compare data across time-points and between volunteers, a crosssample analysis was performed. In this cross sample analysis, the populations identified in a single sample (volunteer 53 s day 10 post infection following stimulation with S. Typhi-infected B-LCL) were applied to all samples. Six unique CD69+ (i.e., recently activated) populations were identified (Figure 9 A). All 6 populations were negative for IL-10 production. MIP-1b production was dependent on the time-point and volunteer. In other words, at those time-points for volunteer 53 s at which intracellular MIP-1b was detected (days 7 and 42 post immunization), MIP-1b was produced by populations 1, 3, 5, and 6. However, for volunteer 54 s, only populations 3 and 5 produced MIP-1b on day 7 post immunization. Population 4 was negative for all cytokines/chemokines measured. Populations 1 and 6 were IL-17A+ (Figure 9). The IL-17A+ populations identified were both multifunctional co-producing IL-2, IFN-c, TNF-a, and/or MIP-1b ( Figure 9). Population 1 was IL-10-IL-17A+ IL-2+ IFN-c+ TNF-a+ MIP-1b+/2, while population 6 was IL-10-IL-17A+ IL-2-IFN-c+ TNF-a+ MIP-1b+/2 ( Figure 9). Figure 9 depicts a representative sample which did not produce MIP-1b (volunteer 53 s day 10 post immunization). The IL-17A+ populations that were identified differed in their production of IL-2.
In addition to identifying multifunctional cells, FLOCK analysis distinguished bright (++) and dim (+) populations of IFN-c and TNF-a based on mean fluorescence intensity (MFI) (Figure 9 A). Brighter intensity of staining indicates higher intracellular cytokine content. Populations 1, 5, and 6 stained brightly for IFN-c, while populations 1 and 6 stained brightly for TNF-a ( Figure 9). These data from unsupervised analysis indicate that IL-17A-producing cells produce high levels of both IFN-c and TNF-a. Figure 10A shows 3-dimensional representation of IL-2 (x-axis), IFN-c (y-axis), and TNF-a (z-axis) for 4 of the CD69+ populations (populations 1, 2, 4, and 6). Each panel shows an approximately 30u rotation around the z-axis. Population 4 (purple) is negative for all measured cytokines, population 2 (lime green) is IFN-c and TNF-a dim, while populations 1 (pink) and 6 (aqua) are IFN-c and TNF-a bright. Populations 1 and 6 are both IL-17A+ but differ in their production of IL-2 as described above (Figures 9A and B and  10A).
The net percentage of positive cells for each population at the 3 time-points, as determined by cross-sample analysis, is shown in Figure 10B. IL-17A+ multifunctional cells were identified at days 7, 10, and 42 post immunization for volunteer 53 s with a peak at day 10 post immunization ( Figure 10B). Volunteer 54 s had low levels of IL-17A+ multifunctional cells on day 7 post immunization, but the predominant response was at day 42 post immunization which correlates with the later peak cytokine production previously demonstrated for volunteer 54 s ( Figure 2C).

Discussion
Here we demonstrate the presence and persistence of multifunctional IL-17A producing S. Typhi-specific CD8+ T cells in response to both S. Typhi-infected autologous B-LCL and HLA-E restricted stimulation. Multifunctional IL-17A+ cells demonstrated multiphasic kinetics and were still detectable one year after immunization. We identified quadruple and quintuple positive CD8+ T EM and T EMRA IL-17A producing cells that co-produce pro-inflammatory cytokines/chemokines IL-2, IFN-c, TNF-a, and/or MIP-1b but not IL-10. These multifunctional populations were confirmed using unsupervised flow cytometric analysis with FLOCK.
IL-17 has been increasingly implicated in host responses against intracellular pathogens [19]. Specifically, the importance of IL-17 in mucosal immune responses to intracellular enteric pathogens has been demonstrated in animal models [20,21]. It was shown that depletion of Th17 cells during simian immunodeficiency virus (SIV) infection results in increased dissemination of Salmonella Typhimurium from the gut [20]. Additionally, antigen-specific IL-17A+ cells were identified in response to Salmonella Enteriditis infection and IL-17A knockout mice had an elevated bacterial burden in the liver and spleen as compared to wild-type mice [37]. Thus, it was of great importance to initiate studies to evaluate whether IL-17A might play a role in protection from S. Typhi. Because the gastrointestinal mucosa is the first point of contact for S. Typhi, mucosal immune responses are likely to play an important role in protection. This is to our knowledge, the first report of IL-17A production in response to S. Typhi, both to S. Typhi-infected autologous targets, as well as HLA-E restricted stimulation. The presence of S. Typhi-specific CD8+ T cells producing IL-17A suggests that these cells may play a role in the mucosal response. CD8+ T cells with characteristics of the Th17 lineage, Tc17, have been described in the setting of chronic hepatitis C virus infection [25]; however, in these studies, stimulation with phorbol myristate acetate (PMA) and ionomycin was used rather than specific antigen. Thus, the results presented in this manuscript are, to our knowledge, the first demonstration in humans of the presence of CD8 + Tc17 cells responsive to antigens from infectious agents.
Multifunctional T cells, i.e., those producing 2 or more cytokines simultaneously, have been shown to produce higher levels of individual cytokines, have enhanced function, and are more likely to correlate with protection from disease, when compared to single cytokine producing cells [29,30,31]. Although T cells co-producing IL-17A with IFN-c or TNF-a in response to PMA/ionomycin stimulation have been identified [38,39], to our knowledge, this is the first study to examine the simultaneous production of IL-17A with 5 other cytokines/chemokines, which play key roles in inflammatory processes, in a human-pathogen specific system. Of note, unsupervised flow cytometric FLOCK analysis showed higher levels of IFN-c and TNF-a being produced by IL-17Aproducing multifunctional cells (i.e., multifunctional IL-17A+ populations are bright for IFN-c and TNF-a). In addition, we

. Typhi in different volunteers. A) CD8+ T EM cells for volunteer 53 s B) CD8+ T EMRA cells for volunteer 53 s C) CD8+ T EM cells for volunteer 54 s D) CD8+ T EMRA cells for volunteer 54 s E) CD8+ T EM cells for volunteer 50 s F) CD8+
T EMRA cells for volunteer 50 s. Increases of .0.5% cytokine positive cells over uninfected targets were found to be statistically significant (P,0.01). * MIP-1b not measured. doi:10.1371/journal.pone.0038408.g004 observed that IL-17A was produced exclusively by multifunctional cells and that the predominant IL-17A+ responses were IL-17A+ IL-2+ IFN-c+ TNF-a+ MIP-1b+/2 as assessed by both supervised (FCOM) and unsupervised (FLOCK) flow cytometric analyses. The co-production of IL-17A with cytokines typically associated with Tc1 responses, suggest that the Tc17 lineage is not as distinct in humans as in vitro and animal studies have previously suggested.
Multiphasic kinetics have previously been demonstrated in response to Ty21a immunization following stimulation in an HLA-E restricted manner [13]. Here we confirm and extend these findings by showing that multiphasic kinetics are also typical of responses to autologous stimulation with B-LCL. Interestingly, despite their bi-or tri-phasic nature, the kinetics of responses to autologous stimulation and HLA-E restricted stimulation differed. HLA-E is a non-classical major histocompatibility (MHC) molecule which is highly conserved [40] and it is expressed ubiquitously on PBMC (at various levels depending on the individual cell types) in combination with classical MHC molecules [41]. Given the differences in kinetics between autologous and HLA-E restricted stimulation, as well as the fact that volunteer 50 s showed responses to autologous stimulation but not to HLA-E restricted stimulation, our results clearly indicate that while HLA-E restricted responses may contribute to the responses seen when PBMC were stimulated with S. Typhiinfected autologous B-LCL, they represent only a portion of these responses. It will be important to confirm these findings using different targets such as autologous blasts. However, these data are supported by the fact that in previous studies, monoclonal antibodies against HLA-E were only able to partially block CTL activity against S. Typhi-infected targets, while monoclonal antibody against a pan-MHC class I marker (W6/32) showed almost complete abrogation of CTL against S. Typhi-infected targets [9]. Moreover, the observations that at different times after immunization there are considerable differences in the effector subsets that respond to autologous and HLA-E restricted S. Typhiinfected cells raises the likely possibility that various effector cells might contribute differently to the host defense at various times after exposure. These observations also point to the importance of the timing of specimen collection following immunization and its impact on the magnitude of effector immune responses which can be measured. Finally, it is important to mention that these data confirm and extend previous observations on the longevity of CMI responses (at least 1 year) elicited following immunization with Ty21a and other attenuated typhoid vaccine candidates [12,13].
The significance of the multiphasic responses observed is not entirely clear; however, it is possible that the decline in the levels of cytokine producing S. Typhi-specific CD8+ T cells at various times could represent these cells homing cells to the gut. Previous studies have, in fact, shown expression of gut-homing molecules on S. Typhi-specific CD8+ T cells [12,17]. While these multiphasic responses have previously been described for HLA-E restricted responses [13], this is the first report of multiphasic kinetics in response to autologous stimulation over the course of 1 year postimmunization with the Ty21a typhoid vaccine. As has previously been shown, the responses following HLA-E restricted stimulation peaked as early as 4-7 days post immunization which is earlier than typically expected for CMI responses [13]. Interestingly, in 2 of the 3 responding volunteers (50 s and 53 s), a peak as early as days 4-7 post immunization was observed in response to autologous stimulation as well. In summary, multifunctional IL-17A+ CD8+ T cells display multiphasic kinetics in response to both classical and non-classical MHC restricted stimulation. These responses persist at least 1 year after immunization. Future studies in which the presence of these multifunctional Tc17 responses are evaluated in the context of clinical outcome following challenge of volunteers with wild-type S. Typhi will provide key information to assess the role of multifunctional cells producing high levels of IL-17A and other cytokines in protection from infection. These studies are likely to contribute to accelerate the development of much needed novel typhoid vaccines.