Parenteral vaccination protects against mucosal Salmonella infection by inducing liver tissue resident memory CD4 T cells

Abstract

Salmonella enterica is a gastrointestinal pathogen that causes a variety of intestinal and systemic clinical disease. Vaccine development for systemic salmonellosis requires a deeper understanding of protective mechanisms and the relative contribution of mucosal and systemic host responses to bacterial elimination. Here, we examined the relative importance of mucosal and systemic immunity in the control of systemic salmonellosis. Delivery of a live-attenuated vaccine strain via a parenteral route did not elicit a substantial mucosal immune response, but was still sufficient to protect mice from systemic Salmonella infection. A prominent feature of the protective systemic response was the establishment of Salmonella-specific tissue resident memory (TRM) CD4 T cells in the liver. Although adoptive transfer of hepatic TRMs was sufficient to protect mice from challenge infection, TRMs from the intestinal lamina propria reduced bacterial loads but failed to fully protect mice from infection. The protective contribution of liver and LP TRMs was partially dependent on the expression of IL-18R and the corresponding ability to elicit noncognate CD4 T cell responses in vivo. Overall, these data suggest that a parenteral vaccination approach that efficiently induces liver TRMs expressing IL-18R should provide robust protection against mucosal or systemic infection with Salmonella.

Introduction

Salmonella is a well-known contaminant of food or fresh produce, regularly causing large outbreaks of gastroenteritis that are reported in the US media.^1–3 However, Salmonella also has a global impact due the ability of certain Salmonella serovars to cause systemic disease. These systemic Salmonella infections usually occur in low-income regions where human-to-human transmission is facilitated by inadequate sanitation and/or limited access to clean water. Salmonella enterica Typhi and S. enterica Paratyphi are 2 human-restricted serovars that cause a clinically indistinguishable systemic disease, referred to collectively as enteric fever (or typhoid).^4,5 Recent estimates suggest that enteric fever afflicts 11 million to 21 million people and causes 148,000 to 161,000 annual deaths.^6,7 The Typhi and Paratyphi serovars spread by human-to-human contact using a fecal/oral route, yet clinical disease is dominated by systemic, rather than intestinal, symptoms.⁸ The systemic nature of Salmonella infection is a consequence of bacterial tropism for the reticuloendothelial system of the spleen, liver, and bone marrow.⁹ A distinct group of non-typhoidal Salmonella (NTS) serovars have evolved similarly to typhoidal strains and cause disseminated infection in young and immune-suppressed individuals in Africa and parts of Asia, a disease referred to as invasive NTS (iNTS).^10,11 Next-generation typhoid conjugate vaccines elicit strong memory B cell responses to the Salmonella Vi capsule polysaccharide (ViCPS), which is effective at reducing enteric fever in endemic areas.^7,12 These vaccines are highly effective and are administered using a parenteral route which effectively induces systemic B cell responses to ViCPS. Unfortunately, 50% of all deaths, and 83% of deaths of children under 5, are caused by Salmonella strains that lack ViCPS expression (Paratyphi and iNTS).¹³ There is, therefore, an urgent need for vaccines that target other Salmonella antigens and can provide protection against iNTS or cross-serovar protection against Typhi and iNTS.

For many years, a major focus of Salmonella vaccine research has been on improving live-attenuated vaccine strains (LVS) that can be administered orally, rather than parenterally.¹⁴ One rationale for using an oral LVS vaccine is the reasonable assumption that mucosal immunity plays a dominant role in mediating protection against oral Salmonella infection. Indeed, many studies have reported Salmonella-specific Ab and T cell responses elicited in the intestine of LVS-immunized mice or humans.^15,16 In mouse models, CD4 Th1 cells are essential for the development of protective immunity, because secretion of IFN-γ is required to control bacterial replication in macrophages.^17–19 However, initial attempts to adoptively transfer lymphocyte-mediated protection from LVS-immunized mice using splenic Th1 cells proved unsuccessful.^9,20 One hypothesis put forward to explain this failure is that tissue resident memory (TRM) CD4 T cells are essential for Salmonella immunity and the spleen does not contain a large population of TRMs.²¹ Indeed, this hypothesis turned out to be accurate, because transfer of liver TRMs from LVS-immunized mice provided robust immunity, yet circulating memory T cells did not.^22–24 These recent findings have some important implications for Salmonella vaccine development because they impose a new requirement that an effective vaccine against systemic disease should aim to generate liver TRMs.¹⁶ It is not yet clear whether there is something unique about liver TRMs or whether Salmonella-specific TRMs from other infected tissues could also be protective.

Given the protective nature of Salmonella-specific liver TRMs it is vitally important to define how these T cells are generated following LVS immunization or infection. We recently reported that certain inflammatory cytokines within hepatic tissue can encourage Salmonella-specific TRM development,²⁵ suggesting that final maturation depends upon local signals within the tissue itself. Additionally, T cell intrinsic T-bet and CD18 expression is required for TRM precursors to mature into liver resident Th1 memory cells.²⁶ Given the oral route of a typical Salmonella infection or LVS administration, it is also important to know whether similar TRMs develop in the intestinal lamina propria (LP) and whether they have protective capacity. Indeed, given the known requirement for liver TRMs, it seems pertinent to ask whether intestinal immunity is required at all. Here, we examine the immune response to LVS immunization after oral or parenteral immunization and find that systemic priming protects against both mucosal and systemic Salmonella infection. Liver TRMs displayed somewhat greater protective capacity than LP TRMs, yet both populations relied on IL-18R expression for robust protection. Overall, these studies show the importance of a vaccine eliciting hepatic TRMs with noncognate functionality and support the feasibility of a parenteral vaccine for all systemic Salmonella infections acquired via a mucosal route.

Materials and methods

Ethics statement

This study was conducted in compliance with the guidelines outlined in the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. The University of California, Davis, holds accreditation from the Association for Assessment and Accreditation of Laboratory Animal Care. All animal experiments received approval from the Institutional Animal Care and Use Committee at the University of California, Davis, under protocol numbers 21869 and 23467.

Mice

C57BL/6 (JAX stock no. 000664), Cd4-cre (JAX stock no. 017336) and B6.129S2-Tcra^tm1Mom/J (JAX stock no. 002115) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) at 6–8 weeks of age and used for experiments at 7–12 weeks old and/or used for establishment of breeding colonies. T cell-specific IL-18R–deficient mice were generated for this study by intercrossing Cd4-cre mice and a new IL-18R^fl/fl mouse line produced by the Mouse Biology Program at the University of California, Davis. All mice were genotyped before use by PCR according to protocols provided by The Jackson Laboratory and housed in specific pathogen-free conditions and cared for in accordance with the University of California, Davis, Institutional Animal Care and Use Committee, and Institutes of Health guidelines.

Bacterial strains and infection

An LVS of S. enterica serovar Typhimurium (BMM50) was developed by the Baumler laboratory (University of California, Davis) through the introduction of a null mutation in the aroA gene of virulent Salmonella strain SL1344. BMM50 was subsequently modified to express a short peptide sequence (EAWGALANWAVDSA) in-frame with OmpC (BMM51).^22,27 Initial pilot experiments verified successful gene targeting, peptide expression, and activation of 2W1S-specific CD4 T cells in vivo. Prior to use, bacteria were streaked on a MacConkey agar plate and individual colonies used to inoculate Luria-Bertani broth (BD), followed by static overnight growth at 37 °C; bacterial concentrations were estimated using a spectrophotometer, with bacteria grown to an OD₆₀₀ between 0.4 and 0.6. Salmonella cultures were subsequently diluted in PBS to concentrations of 5 × 10⁵ CFU/0.2 mL (BMM50), 2.5 × 10⁶ CFU/0.2 mL (BMM51), or 1 × 10³ CFU/0.2 mL (SL1344), with 0.2 mL injected via the lateral tail vein for systemic infection. For oral infection, an appropriate volume of overnight Salmonella culture was centrifuged for 30 min at 4 °C. Supernatant was discarded and bacterial pellets resuspended in 50 mL of water and diluted to appropriate bacterial numbers (1 × 10⁹ CFU) to be used to refill individual water bottles. For oral infection by gavage needle, mice were pretreated with 5% NaHCO₃ solution intragastrically before being infected with Salmonella (5 × 10⁹ CFU/mL) diluted in PBS. The actual concentration of the bacterial dose administered was confirmed by serial dilutions and plating onto MacConkey agar plates in all experiments.

Enumeration of bacteria burden

To assess bacterial colonization in vivo, mice were euthanized via cervical dislocation, and spleen, liver, and intestinal LP from uninfected and infected mice were collected in 1× PBS on ice, homogenized, and brought to final volumes of 2, 5 and 2 mL, respectively. Samples were thoroughly mixed and serial dilutions of the homogenates were plated onto MacConkey agar plates. Following overnight incubation at 37 °C, colonies from serial dilutions were quantified and back-calculated to identify the total colony-forming units present in each organ.

Lymphocyte isolation

Spleens were collected and placed in 2% FBS in 1× PBS. Single-cell suspensions were prepared by pressing the spleens through a 70-μm mesh cell strainer filter unit (08-771-2, Fisher). The suspensions were centrifuged at 2,000 rpm for 5 min at 4 °C, and the supernatant was discarded. To lyse RBCs, the pellet was resuspended in 1 mL of ammonium-chloride-potassium lysis buffer (A1049201, Thermo Fisher) and incubated at room temperature for 3 to 5 min. Lysis was stopped by adding 9 mL of 2% FBS in 1× PBS. The suspensions were centrifuged and decanted as previously described. The pellets were then resuspended in 2% FBS in 1× PBS and cells were quantified either by a Nexcelom Cytometer Auto T4 Bright Field Counter or via a hemocytometer.

Livers were collected and placed in 2% FBS in 1× PBS, and single-cell suspensions were prepared as with spleens. Cellular suspensions were spun at 2,000 rpm for 15 min at room temperature with no brake. Supernatant was discarded and cells were resuspended in 15 mL of 35% isotonic Percoll solution (P1644, Sigma-Aldrich). Cellular suspensions were spun at 2,000 rpm for 20 min at room temperature with no brake. Hepatocytes were gently aspirated and cellular pellets were washed in 40 mL of 2% FBS in 1× PBS. RBCs were lysed, and cells were resuspended in 2% FBS in 1× PBS and quantified.

LP were collected and placed in calcium-magnesium–free (CMF) medium (1× PBS, 1 M HEPES, 5% FBS, 7.5% sodium bicarbonate). Intestinal tissue were kept moist with CMF medium while displacing feces and fat tissue. Intestinal tissue was cut longitudinally and into small pieces and incubated in 10 mL of dithioerythritol (DTE) solution (CMF medium with 10% FBS and 1 mM DTE final) for 30 min at 37 °C with stirring (550 to 750 rpm). Tissue and DTE were transferred to a 50 mL conical tube and vortexed at maximum speed for 1 min then spun at 2,000 rpm for 3 to 5 min at room temperature. Supernatant was aspirated and tissue was washed with DTE solution. Tissue pieces were resuspended in 10 mL of EDTA solution (0.5 M EDTA, 1× PBS, FBS, 1 M HEPES) and incubated for 30 min at 37 °C with stirring (550 to 750 rpm). Pieces then were transferred to a new 50 mL conical tube, spun, and washed with RPMI medium (11875093, Thermo Fisher). Tissue pieces were then resuspended in 10 mL of collagenase solution (0.1 mg/mL collagenase IV, 100 U/mL final, in 5% RPMI medium; C818W76, Thomas Scientific) and incubated for 1 h at 37 °C with stirring (650 rpm) to begin tissue disaggregation. Single-cell suspensions were made by pressing tissue through a 70-μm mesh cell strainer filter unit. Cells were spun as previously described and washed in 40 mL 2% FBS in 1× PBS. Pellets were suspended in 5 mL of 44% Percoll solution (17–0891-02, GE Healthcare) and overlaid onto 3 mL of 67% Percoll solution for a Percoll gradient. Cells were spun at 2,000 rpm for 20 min at room temperature with minimal acceleration and no brake. Lymphocytes from the interface layer were removed from Percoll and washed in 10 mL of 2% FBS in 1× PBS. Cells were resuspended in 2% FBS in 1× PBS and then quantified.

Adoptive transfer of lymphocytes

Liver and LP of LVS-immunized mice were collected and single-cell suspensions were obtained as described above. Liver and LP lymphocytes were counted and normalized to 2 × 10⁶ cells in 200 μL per mouse to be transferred intravenously (IV) into T cell receptor α (TCRα)-deficient recipients.

In vivo stimulation with LPS

Ultrapure LPS from Escherichia coli strain EH100Ra (TLRgrade, Enzo) was diluted in 1× PBS (UltraPure PBS, Gibco) and injected IV at a dose of 10 μg/mouse. Spleens and livers were harvested from uninfected and infected mice 4 h after LPS administration and processed for flow cytometric analysis of cytokine production.

Ab staining and flow cytometry

Single-cell suspensions were made as described under “Lymphocyte isolation” and 1 × 10⁶ cells were incubated with Fc block (culture supernatant from 24G2 hybridoma with 2% mouse serum and 2% rat serum). Cells were incubated with Zombie Yellow (BioLegend) viability dye for 15 min. Cells were washed with 2% FBS in 1× PBS at 2,000 rpm for 5 min at 4 °C and incubated with phycoerythrin-conjugated 2W1S: I-A^b tetramer (NIH Tetramer Facility) in the presence of Fc block for 1 h at room temperature in dark. Cells were washed again and stained with the following antibodies: B220, F4/80, CD11b, CD11c, NK1.1, CD4, CD44, P2RX7, CD69, CD62L, CD11a, CXCR3, and IL-18R. Cells were washed and spun as described above. To access cytokine production and transcription factors, cells were then permeabilized, fixed and stained in intracellular cytokine and transcription factor staining overnight using a Foxp3 intranuclear staining kit (eBioscience). Stained cells were analyzed by flow cytometry using BD LSR Fortessa or BD FACSymphony with appropriate compensation and isotype controls. Flow cytometry data were analyzed with FlowJo software (TreeStar, San Carlos, CA).

Nanobody-mediated inhibition of ARTC2

For flow cytometry analysis of hepatic T cell expression of P2RX7 and transfer experiments, mice received 50 μg of s + 16a nanobody (149802, BioLegend) diluted in 200 μL of PBS, administered 15–30 min prior to organ collection.²⁸

Mouse serum and fecal samples for Ab ELISAs

Groups of C57BL/6 mice were immunized IV with 5 × 10⁵ CFU/0.2 mL (BMM50) or orally with 1 × 10⁹ CFU/water bottle. Blood and fecal pellets were collected 35 days later. Salmonella-specific IgM, IgG2c, and IgA responses were measured using Ab ELISA. In brief, 96-well microtiter plates were coated with heat-killed Salmonella and blocked with 10% FBS/PBS. Serum and fecal samples were added in serial dilutions, followed by incubation with biotin-conjugated anti-mouse IgM, IgG2c, or IgA (BD Biosciences and eBioscience) and HRP-conjugated streptavidin (N100, Thermo Fisher). The plates were developed using an HRP substrate (O-phenylenediamine dihydrochloride, Sigma-Aldrich) and analyzed with an ELISA plate reader (SpectraMax, Molecular Devices).

Statistics

Statistics were performed using Prism version 8 (GraphPad Software). Either a Mann-Whitney U test, 1- or 2-way ANOVA were used. In the presented figures, P values are indicated as follows: *P < 0.05,**P < 0.01,***P < 0.01, and ****P < 0.0001. Graphical figures were made using BioRender.

Results

Mucosal or systemic immunization protect against oral and systemic infection

Although Salmonella is a mucosal pathogen, the relative contribution of mucosal and systemic lymphocytes to host protection remains unclear.²⁹ We decided to explore this issue by immunizing highly susceptible C57BL/6 mice either orally or IV with an AroA-deficient LVS of Salmonella. As expected,³⁰ oral immunization fully protected mice from subsequent oral challenge with a virulent strain (SL1344) that usually causes a fatal infection in naïve mice (Fig. 1A). Oral immunization was also effective at protecting almost all mice against an IV-introduced systemic infection using the same virulent strain (Fig. 1A), thus demonstrating that mucosal immunization can induce effective systemic immunity.

Figure 1.

Mucosal or systemic immunization can protect against oral or systemic infection. C57BL/6 mice were orally (A, C, and D) or IV (B, E, and F) immunized with an LVS of Salmonella. All groups were challenged with virulent Salmonella SL1344 orally and IV at 45 d after infection and protection was determined by survival (A and B) or bacterial burdens of spleen (C and E) and liver (D and F). Data are representative of 2 experiments with 3 to 4 mice in each group. Data are shown as mean ± SEM and analyzed by unpaired Student t test (C to F). **P ≥ 0.001, ***P ≥ 0.0001.

Next, we examined the protective effect of parenteral immunization using the same AroA-deficient LVS-Salmonella. As expected, immunization of C57BL/6 mice via the IV route provided robust protection against subsequent systemic (IV) infection with Salmonella SL1344 (Fig. 1B). Perhaps more surprisingly, IV immunization was also highly effective at protecting mice from oral challenge with Salmonella, demonstrating that parenteral immunization can protect effectively against mucosal challenge. Indeed, when examined more carefully in subsequent experiments in which bacterial burdens were enumerated, IV immunization reduced bacterial burdens in the spleen and liver, irrespective of whether immunized mice were challenged using a systemic (Fig. 1C and D), or oral route (Fig. 1E and F). Thus, parenteral immunization using an LVS strain provides robust protection against a mucosal Salmonella infection.

Systemic immunization induces systemic and mucosal memory T cells

Ab and CD4 T cells each play distinct roles in immunity to Salmonella^31–33; therefore, we examined these responses after oral or IV immunization. Mice immunized orally or IV with LVS-Salmonella displayed similar IgM and IgG2a responses to Salmonella antigens (Fig. 2A and B). A small Salmonella-specific IgA response was detectable in serum after oral immunization, but no detectable secretory IgA was evident in fecal pellets from either group of immunized mice. Thus, LVS immunization elicits similar serum IgM and IgG responses and low IgA, irrespective of which route of immunization is used.

Figure 2.

LVS immunization elicits similar serum IgM and IgG responses, and low IgA response, irrespective of route of immunization. C57BL/6 mice were immunized orally or IV with Salmonella enterica Typhimurium and, 35 d later, serum and fecal pellets were collected to determine Ab responses by ELISA. Line graphs show mean absorbance of 4 mice per group.

Next, we examined the induction of Salmonella-specific CD4 memory T cells in systemic and mucosal tissues after IV immunization. Salmonella-specific memory CD4 T cells were identified using 2W1S MHC class II tetramers after immunization with AroA-deficient Salmonella expressing 2W1S.^22,25,34 Again, IV immunization with LVS-Salmonella led to the generation of memory CD4 T cells in the spleen, liver, and LP, as defined by an increased percentage and absolute number of tetramer-positive CD44⁺ CD4 T cells (Fig. 3A–C). In the liver and LP, the majority of memory CD4 T cells expressed the surface molecules CD69 and P2RX7 (Fig. 3D–F), indicating TRM development. It was notable that total number of Salmonella-specific TRMs in the LP was much less than in the liver (Fig. 3D–F). Despite the expectation that TRM formation might be greatly enhanced in the LP after oral immunization, the number of LP memory T cells remained low and was not statistically different from that observed with IV immunization (Fig. 3G and H). However, the few Salmonella-specific memory cells detected in the LP after oral or IV immunization almost uniformly expressed CD69 and P2RX7 (Fig. 3I–K). Thus, irrespective of the immunization route used, LVS immunization strongly induces hepatic TRM T cells, but only induces a small population of TRMs within the LP.

Figure 3.

Systemic immunization induces systemic and mucosal memory T cells. C57BL/6 mice were immunized with Salmonella-2W1S–LVS IV (A–K) or orally (G–K) and, 45 d later, spleen, liver, and LP were collected and analyzed by flow cytometry. Immunization-specific CD4 T cells were identified by 2W1S-tetramer staining (A–C, G) and TRM were identified as CD69⁺P2RX7⁺ tetramer-positive CD4 T cells (D–F, I–K). *P ≥ 0.01, **P ≥ 0.001, ***P ≥ 0.0001, ****P ≥ 0.0001. OG, oral gavage.

Liver TRM T cells are more protective than intestinal TRM T cells

The results thus far demonstrated that liver TRMs are a major fraction of the CD4 T cell memory response after LVS-Salmonella immunization, irrespective of the immunization route used. We previously reported that adoptive transfer of Salmonella-specific TRMs to naïve mice provides substantial protection from challenge, as long as treatment with the s + 16s nanobody is used to prevent TRM cell death.^22,23,26 Thus, we compared the protective capacity of Salmonella-specific TRMs recovered from either the liver or the LP.

In initial experiments to examine their homing potential, T cells isolated from the liver or LP of LVS-immunized mice were examined in vivo after transfer into TCRα-deficient recipients (with and without s + 16a). Transferred liver TRMs were detected in the spleen and LP of recipient mice, but, overall, these cells displayed a preference for the liver, especially when protected from NAD-mediated cell death using s + 16a (Fig. S1A and B). In marked contrast, transferred LP TRMs were detected in all tissues of recipient mice irrespective of s + 16a (Fig. S1C and D). Thus, liver TRMs seem to display a preference to return to their tissue of origin, which has previously been shown to depend on T-bet regulation of surface CD18.²⁶

Next, we examined the protective effect of liver versus LP TRM. Recipients of liver or LP TRMs were challenged IV with attenuated bacteria or orally with wild-type bacteria to monitor protection using each infection route (Fig. 4A). Mice with no T cell transfer at all had uniformly high levels of bacteria in the spleen and liver, whether challenged IV or orally (Fig. 4B–E, no transfer). As a positive control, mice immunized with LVS-Salmonella displayed a > 1,000-fold reduction in bacterial burdens in both organs (Fig. 4B–E, immunized). The adoptive transfer of LP TRMs lowered bacteria counts in the liver and spleen in mice challenged IV; however, this protective effect was only detected in the spleen after oral challenge (Fig. 4B–E, LP transfer). Interestingly, the transfer of liver TRMs provided greater protection than LP TRMs, but only when mice were challenged via the IV route (Fig. 4B–E). In contrast, the protection from oral infection was broadly similar irrespective of whether recipient mice had received liver or LP TRMs.

Figure 4.

Liver TRM T cells are more protective than intestinal TRM T cells. C57BL/6 mice were immunized with Salmonella-2W1S IV and, 45 d later, received 50 µg S + 16a or PBS by IV injection 15 min before harvest of T cells isolated from the liver and LP for adoptive transfer into TCRα-deficient recipients. Recipients were challenged IV with attenuated Salmonella (A–C, F) or virulent Salmonella orally (A, D, E, and G) 24 h after adoptive transfer. Significance was calculated by 1-way ANOVA; data are reported as mean + SEM.

To determine whether these differences in bacterial counts equated to longer survival times for mice with transferred TRMs, we transferred liver or LP TRMs and subsequently examined survival after IV or oral challenge. Irrespective of the route of challenge, the transfer of liver TRMs provided substantial protection to greater than 75% of all infected mice (Fig. 4F and G). In contrast, the transfer of LP TRMs extended survival time but ultimately failed to protect any recipient mice, whether they were challenged IV or orally (Fig. 4F and G). Thus, in this head-to-head analysis experiment, liver TRMs were more protective than LP TRMs, irrespective of whether mucosal or IV challenge routes were used.

Liver TRM T cells display robust noncognate responses

Salmonella-specific CD4 T cells protect the host via the secretion of IFN-γ, which activates Salmonella-infected macrophages.^18,35 The secretion of IFN-γ by Th1 cells can also be amplified by local noncognate (i.e. non-TCR) signals, especially by signaling through the IL-18R.^36–38 We initially noted that IL-18R expression was slightly higher on liver than on LP TRMs (Fig. 5A), suggesting that this cytokine receptor might be responsible for enhancing their protective capacity. To examine this issue, we generated an IL-18Rα–floxed mouse and crossed this line to CD4-Cre, thus generating mice lacking IL-18R expression on T cells (Fig. 5A). As expected, CD4 T cells from CD4/IL-18R–deficient mice produced less IFN-γ in response to noncognate activation in vivo (Fig. 5B). Similarly, these mice had modestly elevated bacterial burdens in the spleen and liver after primary infection with Salmonella (Fig. 5C and D), demonstrating the importance of this IFN-γ amplification loop to full protection. Despite having a deficiency in noncognate responsiveness, CD4/IL-18R–deficient mice still eventually resolved primary infection and developed a similar number and percentage of tetramer-positive CD44⁺ memory CD4 T cells, compared with CD4Cre-negative control mice (Fig. S2A to C). The formation of liver TRMs was also similar in CD4/IL-18R–deficient mice compared with controls (Fig. S2D to F), demonstrating that expression of IL-18R itself is not essential for hepatic CD4 TRM development. This capacity to generate hepatic TRMs in the absence of IL-18R allowed us to directly examine whether IL-18R was essential for the protection mediated by liver or LP TRMs (Fig. 6A).

Figure 5.

Liver TRM T cells display robust noncognate responses. (A) C57BL/6 mice were immunized with Salmonella-2W1S–LVS IV and, 45 d later, spleen, liver, and LP were collected and analyzed by flow cytometry to examine IL-18R surface marker expression. n = 4 for all experiments; data are representative of 2 experiments. Significance was calculated by 1-way ANOVA and data are reported as mean + SEM. ****P ≥ 0.0001. (B–D) CD4/IL-18R–deficient mice and littermate control were infected with LVS-Salmonella. Two weeks later, infected mice were injected IV with 10 µg LPS, and spleens were collected 4 h later to examine IFN-γ production by Th1 cells by flow cytometry (n = 6) (B). Bacterial loads in the spleen and liver of individual mice were determined at day 35 after infection (n = 5) (C and D). (B) Significance was calculated by 2-way ANOVA; data are reported mean + SEM. (C and D) Data are reported as mean ± SEM and analyzed by unpaired Student t test. *P ≥ 0.01. SSC, side scatter.

Figure 6.

Expression of IL-18R contributes to protection mediated by intestinal memory CD4 T cells. CD4/IL-18R–deficient mice and littermate controls were immunized with LVS-Salmonella and, 45 d later, T cells isolated from the liver (B) and LP (C) were adoptively transferred into TCRα-deficient recipients (A). Recipients were challenged IV with attenuated Salmonella after adoptive transfer to determine protective capability.

Consistent with these data, the adoptive transfer of Cre-/IL-18^fl/fl liver TRMs protected greater than 80% of recipient mice from Salmonella infection (Fig. 6B). However, the adoptive transfer of liver TRMs lacking IL-18R expression provided somewhat lower protection against infection (Fig. 6B). Thus, the ability to respond to noncognate stimuli is an essential component of liver TRM secondary protection against Salmonella infection, in agreement with this protective role during primary infection (Fig. 5). As observed with the adoptive transfer experiments, the transfer of Cre-/IL-18^fl/fl LP TRMs provided less protection than did hepatic TRMs (Fig. 6C): LP TRMs were only able to fully protect 60% of recipients. Interestingly, this lower protective capacity of LP TRMs was further reduced in the absence of IL-18R (Fig. 6C), demonstrating that LP TRMs also require IL-18R for their more modest protective capacity. Thus, a major aspect of the protective capacity of both liver and LP TRMs is the ability to enhance IFN-γ production via detection of local inflammatory signals.

Discussion

Immunity to oral pathogens is often discussed in the context of the protective host response at the intestinal mucosa.^39,40 Indeed, mouse models of Salmonella infection have been used to understand the basic function of M cells, Peyer’s patches, IgA, and mucosal lymphocyte responses.^41–44 However, although Salmonella obviously uses an intestinal route of entry into the host, the requirement for mucosal memory responses to protection against infection is less clear.²⁹ This issue is important because approximately 200,000 people succumb to systemic salmonellosis annually after ingestion of contaminated food or water.^7,45 Furthermore, effective vaccines are not yet available for the majority of Salmonella infections.¹³ It has been known for decades that oral immunization of mice using attenuated Salmonella provides robust protection against infection.³⁰ This finding has led to the development of many LVS of Salmonella that can be orally administered and that are moderately effective when used as vaccines.¹⁵ One underlying assumption of this vaccine approach is that oral vaccine delivery is preferable because it can stimulate sufficient mucosal immunity to protect against a subsequent oral infection.^14,15 However, our study challenges this notion, because systemic administration of LVS Salmonella also effectively protected against mucosal infection without the induction of strong mucosal immune memory. It is important to note that these data do not necessarily mean that the mucosal immune response lacks capacity to protect against oral infection; rather, a requirement for protection of the intestine itself is less obvious than would be expected for a mucosal pathogen.

When examining the induction of mucosal and systemic immune responses following LVS immunization, we found that TRMs were induced in both the liver and LP; however, the frequency of these cells was noticeably higher in the liver. This result fits well with a model in which the majority of T cell clonal expansion occurs in the spleen, before the expanded memory precursors take up residence in tissue sites. Because there is extensive bacterial replication in the liver (but not in the LP) during systemic Salmonella infection, the higher antigen load within the liver may favor maturation of TRMs within this tissue. Furthermore, the replication of LVS Salmonella in the liver most likely induces the necessary inflammatory signals to encourage TRM formation.²⁵

Adoptive transfer of liver TRMs to immune-deficient mice provided substantial protection against infection, whereas transfer of LP TRMs was less effective. This is admittedly not a perfect comparison, because each isolated population likely has different homing properties that could influence their protective capacity within a new host. However, when equal numbers of TRMs were transferred, both TRM populations reduced bacterial replication, demonstrating that both were functional in vivo. However, only the transfer of liver TRMs dramatically enhanced overall survival, suggesting that this population has greater protective capacity in vivo. One interesting aspect of this transferred protection is the requirement for expression of IL-18R. Several groups have reported the importance of IL-18 and noncognate activation of CD4 T cells in immunity to Salmonella,^36,37,46,47 but to our knowledge, this is the first time IL-18R has been shown to be essential for the full protective capacity of memory CD4 T cells. The absence of IL-18R expression also reduced the protective capacity of LP TRMs, thus demonstrating the vital importance of this receptor in bacteria-specific memory Th1 responses, irrespective of tissue source. Noncognate stimulation of CD8 T cells has been described for viral infections,⁴⁸ where effector T cell responses are often amplified by local inflammation. It remains unclear why noncognate activation is so vital for Salmonella clearance, but it seems possible that Salmonella inhibitory effects on MHC class II allow for the evasion of Th1 cell killing via cognate recognition.^49,50 Studies examining other intramacrophage bacteria have shown the essential nature of cognate activation by the T cell receptor.⁵¹ However, studies using bacterial mutants that are unable to inhibit MHC class II expression on macrophages will be needed to resolve whether this is a mechanistic explanation for a requirement for noncognate Th1 cell responses.⁵²

Overall, our study has implications for the design of vaccines for systemic salmonellosis, especially those that would attempt to protect against multiple serovars. Basically, we found that induction of hepatic TRMs is a critical component to the control of systemic replication of Salmonella, whereas induction of mucosal immune responses is dispensable. Indeed, this finding is logical because bacterial replication primarily occurs in the liver rather than the LP. Even if mucosal immune responses could be induced that had the potential to control the majority of bacteria upon intestinal entry, a small number of bacteria might still reach the liver, where they could multiply and cause systemic symptoms. Vaccines for systemic Salmonella may be more effective if they focus on parenteral administration, maximize the expansion of Th1 cells in lymphoid tissues, and encourage liver memory residency.⁵³ Studies are planned to develop a parenteral Salmonella vaccine that can elicit strong liver TRM responses to test this possibility.

Author contributions

Alana Nguyen (Data curation [Lead], Formal analysis [Equal], Investigation [Equal], Methodology [Equal], Writing—review & editing [Equal]), Kevin Fong (Formal analysis [Supporting], Investigation [Supporting], Methodology [Supporting]), Marietta M Ravesloot-Chavez (Formal analysis [Supporting], Investigation [Supporting], Methodology [Supporting]), Vy Pham (Formal analysis [Supporting], Investigation [Supporting], Methodology [Supporting]), Jesica Calderon (Formal analysis [Supporting], Investigation [Supporting], Methodology [Supporting]), and Stephen J. McSorley (Conceptualization [Lead], Formal analysis [Lead], Funding acquisition [Lead], Project administration [Lead], Resources [Lead], Supervision [Lead], Writing—original draft [Lead], Writing—review & editing [Lead])

Supplementary material

Supplementary material is available at The Journal of Immunology online.

Funding

This study was supported by National Institutes of Health funding (grants R01AI139410, R21AI187632, R21AI196078, and T32AI060555 to S.J.M.) and the Eugene Cota Robles Award and Robert Emrie Smith Memorial Research Fellowship (to A.N.).

Conflicts of interest

None declared.

Data availability

All data will be made freely available to any academic researcher upon request.