Lung CD4 Tissue-Resident Memory T Cells Mediate Adaptive Immunity Induced by Previous Infection of Mice with Bordetella pertussis

Whooping cough (pertussis) is an infectious disease of the respiratory tract caused by the Gram-negative bacterium Bordetella pertussis. It is especially serious in young children, pregnant women, the elderly, and immune-compromised individuals, and it can be fatal in infants. The incidence of pertussis is increasing worldwide, with recent outbreaks reported in many developed countries (1). This has been linked with evolution of the bacteria and ineffective or waning immunity induced with current acellular pertussis (aP) vaccines (2, 3). The duration of immunity is greater following infection compared with that induced by immunization with aP vaccines, and this may reflect the superior induction of immunological memory by natural infection. We (4–6) and other investigators (7) have demonstrated that infection with B. pertussis induces peripheral Th1 cells in mice and in children. Studies in a mouse model have demonstrated that Th1 cells, together with Th17 cells, confer immunity to B. pertussis (8–10). In contrast, aP vaccines, which are less protective than whole-cell vaccines, induce Th2 cells and strong Ab responses (5, 8). However, Ab responses and protective immunity wane rapidly in children, even after five doses of aP (3). The role of memory T and B cells and local T cells in the respiratory tract in mediating immunity to B. pertussis has received less attention (11–13).

Memory T and B cells induced by vaccination or previous infection provide rapid and effective protective immunity against infection or reinfection. Memory T cells have classically been subdivided into central memory T (T_CM) cell and effector memory T (T_EM) cell subtypes. T_CM cells express l-selectin and CCR7 and migrate to secondary lymphoid organs, where they can differentiate into effector T cells in response to Ag stimulation. T_EM cells migrate to inflamed peripheral tissues and display immediate effector function. A recently identified subpopulation of memory T cells that resides in peripheral tissues and does not recirculate has been termed tissue-resident memory T (T_RM) cells (14). T_RM cells that express CD69 or CD69 and CD103 (α_Eβ₇ integrin) provide a first line of defense against infection at mucosal surfaces, responding rapidly without a need for recruitment of T cells from the circulation. T_RM cells that reside in the epithelium (or epithelial tissues) of a mucosal site are specific for pathogens previously encountered at that mucosal tissue (15, 16). CD8 T_RM cells have been described in the lungs, skin, and vagina of mice following viral infection (17–19). There is a more limited number of reports on CD4 T_RM cells, although lung-resident CD4 T cells have been shown to play a role in protection against influenza virus infection of mice (20). Furthermore, a subset of CD4 T cells that homes to and resides in the lung parenchyma provided superior protection against Mycobacterium tuberculosis infection than did circulating intravascular CD4 T cells (21).

In this study, we used an inhibitor of lymphocyte migration, FTY720 (fingolimod), to examine the role of migratory T and B cells into the lungs and the role of memory T cell subtypes in the clearance of primary and secondary B. pertussis infections in mice. FTY720 inhibits the sphingosine-1-phosphate receptor pathway, thereby impairing the trafficking of lymphocytes from lymph nodes (LNs) to tissues (22). FTY720 is an orally active immunomodulatory drug licensed to treat relapsing forms of multiple sclerosis; however, the results of clinical trials showed an increased incidence of respiratory tract infections in FTY720-treated patients (23). Studies in mice have shown that chronic treatment with FTY720 impairs the clearance of Citrobacter rodentium (24) and influenza virus (20). However, there have also been reports of positive impacts of sphingosine-1-phosphate receptor agonists, with AAL-R preventing inflammation or cytokine storms during the effector phase of infection with influenza virus (25) or B. pertussis (26).

The objective of this study was to determine whether CD4 T_RM cells accumulate in the lungs during infection and/or reinfection with B. pertussis and whether they played a role in protective immunity. We found that treatment with FTY720, which inhibited migration of lymphocytes from LNs to lungs, significantly impaired clearance of a primary infection. Cells with T_EM and T_RM phenotypes expanded in the lungs during B. pertussis infection, and this was not affected by treatment with FTY720. Moreover, bacteria were rapidly cleared from the lungs following reinfection of FTY720-treated and untreated convalescent mice, and this was associated with further local expansion of T_RM cells. In addition, we showed that lung T_RM cells adoptively transferred from convalescent mice into naive irradiated mice promoted clearance of B. pertussis infection from recipient mice. Our findings point to an important role for CD4 T_RM cells in mediating rapid protective immunity induced by previous infection with B. pertussis.

C57BL/6 (8-wk-old) mice were obtained from Harlan Laboratories U.K. or the Comparative Medicine Unit, Trinity College Dublin and housed in a specific pathogen–free facility. All animal experiments were conducted in accordance with the recommendations and guidelines and under licenses approved by the Health Products Regulatory Authority of Ireland in accordance with protocols approved by the Trinity College Dublin Animal Research Ethics Committee.

B. pertussis bacteria were grown from a frozen stock on Bordet Gengou plates containing glycerol and horse blood (Cruinn) at 37°C. After 3 d of culture, the bacteria were collected in supplemented Stainer–Scholte medium and cultured overnight at 37°C in a shaking incubator at 220 rpm. Bacteria were centrifuged and resuspended in 1% casein solution, and the OD was measured at 600 nm. B. pertussis infection of C57BL/6 mice was performed by aerosol challenge (BP338 strain; 1 × 10⁹ CFU/ml) administered using a nebulizer (PARI TurboBOY SX) over 10 min (13). The course of infection was followed by performing CFU counts on lung homogenates at intervals postinfection (p.i.), as described (13).

FTY720 (Santa Cruz Biotechnology) was administered to mice orally in the drinking water at a concentration of 0.3 mg/kg/d for a period of 10 d before infection and until the cessation of the experiment.

To discriminate blood-borne circulating cells from lung-localized cells, we used a well-described approach in which anti-mouse PE-CD45 Ab (eBioscience) was administered i.v. to mice 10 min before they were euthanized and lungs were harvested (27).

Lung tissue was chopped and digested with collagenase D (1 mg/ml; Roche) and DNase I (10 μg/ml; Sigma-Aldrich) for 1 h at 37°C with agitation. Next, lungs or spleens were passed through a 40-μm cell strainer to a obtain single-cell suspension, followed by RBC lysis. The cells were incubated with CD16/CD32 FcγRIII (1:100) to block IgG Fc receptors. Cells were incubated with LIVE/DEAD Aqua (Invitrogen), followed by surface staining with fluorochrome-conjugated anti-mouse Abs for various markers. To detect cytokines, cells were stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of brefeldin A (5 μg/ml) for 4 h at 37°C. The following surface Abs were used: CD45R-BV605, CD3-BV650, CD44-BV605 (BioLegend), CD62L–PE–CF594, CD103-BV785 (BD Biosciences), CD45R–Alexa Fluor 700, CD4–allophycocyanin–eFluor 780, CD69-FITC, CCR7-allophycocyanin, CD8–PE–Cy7, CD3–allophycocyanin–eFluor 780, CD4–Alexa Fluor 700, CD49b-allophycocyanin, and CD8–PE–Cy7 (eBioscience). For detection of intracellular cytokines, cells were fixed in 2% PFA and permeabilized with 0.5% saponin (Sigma-Aldrich, Ireland), followed by staining with IL-17A–V450 and IFN-γ–PE–CF594 (BD Biosciences). The Foxp3/Transcription Factor Staining Buffer Set (eBioscience) was used, according to the manufacturer’s protocol, to detect expression of the proliferation marker Ki67 in memory T cells. Fluorescence minus one or nonspecific isotype Abs were used as controls. Flow cytometric analysis was performed on an LSR Fortessa, and data were acquired using Diva software (BD Biosciences). The results were analyzed using FlowJo software (TreeStar).

Lung mononuclear cells were pooled from four untreated and FTY720-treated mice and prepared as described above, followed by cell separation over continuous 30% Percoll gradient (GE Healthcare). Spleens from untreated and FTY720-treated mice were passed through a 40-μm cell strainer to obtain a single-cell suspension. CD4 T cells from lung or spleen were sorted by negative selection (Miltenyi Biotec). Spleen cells or purified CD4 T cells from lung or spleen mixed with APCs (irradiated spleen cells) were cultured with filamentous hemagglutinin (FHA; 2 μg/ml), sonicated B. pertussis (sBp; 10 μg/ml), heat-killed B. pertussis (hkBp; equivalent to 10⁷ CFU/ml), or medium only. After 3 d, IFN-γ and IL-17A concentrations were quantified in supernatants by ELISA.

Lung mononuclear cells were pooled from 15 naive or 15 convalescent mice and prepared as described above, followed by cell separation over continuous 30% Percoll gradient (GE Healthcare). Spleens from naive and convalescent mice were pressed through a 40-μm cell strainer to obtain a single-cell suspension. CD4 T cells were sorted by negative selection (Miltenyi Biotec). Naive mice were irradiated (5 Gy) 1 d before cell transfer using a Gammacell irradiator. A total of 2 × 10⁶ CD4 cells was transferred i.v. 1 d before challenge. PBS-injected mice served as controls.

Statistical analysis was performed using GraphPad Prism. Data were analyzed using one- or two-way ANOVA, followed by the multiple comparison tests, or a two-tailed unpaired t test, as appropriate. Data are expressed as mean with SEM or SD and were deemed statistically significant when p < 0.05.

Evaluation of CFU in the lungs after aerosol challenge of naive mice with B. pertussis showed that the bacterial load peaked on day 7, and the bacteria were cleared by day 42 p.i. In contrast, convalescent mice (day 60 p.i.) rapidly cleared the B. pertussis infection from the lungs following reinfection, with complete clearance by day 7 (Fig. 1A). The clearance of B. pertussis from the lungs was associated with an increase in the number of CD4 T cells in the lungs (data not shown). To examine the accumulation of CD4 T_RM cells and their possible role in the clearance of B. pertussis infection from the respiratory tract, we monitored the expression of the tissue-retention markers CD69 and CD103 on total CD4 T cells during the course of primary infection and following reinfection with B. pertussis. To discriminate blood-borne circulating leukocytes from lung-retained leukocytes, we used a validated approach (27) in which a fluorescently labeled anti-CD45 Ab was administered i.v. to mice 10 min prior to sacrifice. Circulating cells become labeled with the Ab (CD45⁺), whereas the Ab cannot penetrate the tissue to stain the lung-resident cells and, therefore, they remain unstained (CD45⁻). When examined 21 d after B. pertussis challenge, lung-resident CD4 T cells expressed the tissue-resident memory cell markers CD69 and/or CD103. Moreover, CD4 T_RM cells were still elevated in the lungs of convalescent mice at day 60 p.i. (Fig. 1B) and persisted until at least day 120 p.i. (data not shown). Interestingly, CD4 T cells with a tissue-resident memory phenotype expanded significantly (>6-fold) after reinfection (Fig. 1C), suggesting that they may play an important role in the rapid clearance of B. pertussis from the lungs following reinfection.

To examine the role of local tissue-resident CD4 T cells versus peripheral/circulating CD4 T cells in protection against B. pertussis infection, FTY720 was used to inhibit the migration of lymphocytes from the LNs to the lungs (22). Mice were treated with FTY720 orally for 10 d before challenge and over the course of infection with B. pertussis. Treatment with FTY720 significantly exacerbated infection and delayed clearance of B. pertussis from the lungs (Fig. 2A). The infection peaked on day 8 after challenge; although untreated mice had cleared the infection by day 42, FTY720-treated mice were still infected on day 60 and had significantly higher CFU in the lungs on days 35, 42, and 60 p.i. Using the i.v. labeling approach described above, we analyzed circulating and lung-resident CD4 T cells during the course of infection in FTY720-treated and untreated mice. CD4 T cells were markedly reduced from the circulation of FTY720-treated mice but not untreated animals (Fig. 2B). In contrast, lung CD4 T cells expanded significantly by day 14 p.i. and remained elevated over the course of infection in untreated and FTY720-treated mice. However, untreated mice had significantly more CD45⁻CD4 T cells in the lungs on days 14 and 35 p.i. compared with FTY720-treated animals (Fig. 2B).

Flow cytometric analysis of lung mononuclear cells from untreated and FTY720-treated naive mice revealed a significant reduction in circulating (CD45⁺) and lung-resident (CD45⁻) B and CD4 T cells in FTY720-treated mice compared with untreated mice, suggesting that most of these cells transiently penetrate the tissue and are significantly reduced when migration from the LNs is blocked (Fig. 2C). Furthermore, there was a reduced frequency of circulating B and CD4 T cells in FTY720-treated mice compared with untreated mice at day 21 p.i. Consistent with this, B. pertussis–specific serum IgG2c, the dominant isotype in B. pertussis–infected C57BL/6 mice, was substantially reduced in mice treated with FTY720 during primary infection (Supplemental Fig. 1). However, although the frequency of lung B cells was persistently suppressed in all FTY720-treated mice, the frequency of lung-resident CD4 T cells increased compared with naive FTY720-treated mice (Fig. 2C). The increase in the number of CD4 T cells in the lungs p.i., despite constant treatment with FTY720, suggested a local expansion of cells that were present in the lungs before the infection, or it may reflect the infiltration of CD4 effector T (T_eff) cells into the lungs during the effector phase of the immune response.

To address these possibilities, we carried out flow cytometry on lung-infiltrating CD4 T cells in FTY720-treated and untreated B. pertussis–infected mice using Abs specific for CD62L and CD44 to discriminate among naive T (T_N), T_CM, and T_eff/T_EM cell phenotypes and using Abs specific for CD69 and CD103 to examine T_RM cells. It has been demonstrated that FTY720 preferentially retains T_N and T_CM cells, but not cells with a T_EM-like phenotype, in the LNs (28). Infection of mice with B. pertussis was associated with an enhancement of the number of CD44⁻CD62L⁺ T_N cells_, CD44⁺CD62L⁺ T_CM cells, and CD44⁺CD62L⁻ T_EM-like cells in the circulation that peaked on days 14 and 42 postchallenge, and each memory T cell population was reduced to background levels following treatment with FTY720 (Fig. 3). There was a small increase in the number of CD44⁻CD62L⁺ T_N cells and CD44⁺CD62L⁺ T_CM cells in the lungs during infection with B. pertussis, and this was reduced to background levels in FTY720-treated mice. In contrast, there was a significant increase in the numbers of CD44⁺CD62L⁻ T_EM-like cells in the lungs during infection with B. pertussis, and these cells were not significantly reduced by treatment with FTY720 (Fig. 3).

We next examined the possibility that T_EM-like cells in the lung had a T_RM phenotype and that local expansion of these cells may explain the increased number of CD44⁺CD62L⁻ T_EM-like cells. We found that a significant number of lung-infiltrating CD4 T cells had a T_RM cell phenotype (CD44⁺CD62L⁻CD69⁺ or CD44⁺CD62L⁻CD69⁺CD103⁺). The frequency of CD44⁺CD62L⁻CD69⁺ and CD44⁺CD62L⁻CD69⁺CD103⁺ T_RM cells was similar in untreated and FTY720-treated mice during the course of infection (Fig. 3B). Furthermore, the absolute number of CD44⁺CD62L⁻CD69⁺ (Fig. 3A) and CD44⁺CD62L⁻CD69⁺CD103⁺ (data not shown) T_RM cells in the lungs was not affected by treatment with FTY720. CD4 T cells with a T_RM-like phenotype accounted for almost all CD4 T cells in the lungs of FTY720-treated mice, suggesting that this lung-retained population of CD4 T cells expanded in situ in response to B. pertussis infection.

When taken together with our previous studies showing that Th1 and Th17 cells mediate natural immunity to B. pertussis infection (8, 9), the data presented in Fig. 3 provide indirect evidence that lung-infiltrating T and B cells play an important role in the clearance of bacteria from the respiratory tract. To examine whether the infiltrating T cells were Th1- or Th17-type cells, we performed intracellular cytokine staining for IL-17 and IFN-γ on CD4 T cells from the lungs of infected mice. IFN-γ– and/or IL-17–secreting CD4 T cells were detected in the lungs during infection with B. pertussis on days 28 and 60 p.i. and, surprisingly, the frequency of these Th1 and Th17 cells was enhanced in FTY720-treated B. pertussis–infected mice (Fig. 4A). The absolute number of IFN-γ– or IL-17–secreting CD4 T cells was similar in FTY720-treated and untreated mice on day 28 of infection, and it was greater, although not significantly, in the FTY720-treated mice 60 d postchallenge with B. pertussis (Fig. 4B). These findings demonstrate that, although FTY720 suppresses infiltration of total CD4 T cells into the lung, the frequency and absolute numbers of CD4 T cells that secrete IFN-γ or IL-17 are not impaired by FTY720 treatment. It is possible that local Ag-specific Th1 or Th17 cells expand in the lung late during infection with B. pertussis and/or that FTY720 has selective effects on different subtypes of T cells. Interestingly, the frequency of IL-17–secreting CD4 T cells was higher than that of IFN-γ–secreting CD4 T cells (Fig. 4A, 4B), suggesting preferential expansion of Th17 cells in the lungs.

We next examined Ag-specific Th1- and Th17-type responses in the spleen and lung in FTY720-treated or untreated convalescent mice 60 d after initial challenge with B. pertussis. Total spleen cells or purified lung CD4 T cells and APCs (irradiated spleen cells) were stimulated with hkBp, sBp, or the purified B. pertussis Ag, FHA. Significant concentrations of IFN-γ and IL-17 were detected in the supernatants of spleen cells and lung CD4 T cells stimulated with B. pertussis Ags. Treatment with FTY720 appeared to reduce the responses in the spleen but not in the lungs (Fig. 4C). It should be noted that, although the in vitro response of the lung-infiltrating CD4 T cells was similar in FTY720-treated and untreated mice, the numbers of cells that infiltrated into the lungs was lower in FTY720-treated mice. Furthermore, when we examined the responses of purified CD4 T cells from the spleen (with added APCs), we found that IFN-γ and IL-17 production was significantly greater in mice treated with FTY720 (Supplemental Fig. 2). This may reflect selective retention of activated/effector T cells, which are not affected by FTY720. Collectively, the data suggest that B. pertussis–specific Th1 cells, and especially Th17 cells, are present at high numbers in the lungs during infection with B. pertussis and that this may reflect local expansion, as well as recruitment from peripheral lymphoid tissue.

We next investigated the role of CD4 T_RM cells that expand in the lungs during infection with B. pertussis in the protection against secondary infection in convalescent mice. Convalescent mice were treated with FTY720 starting from day 10 before secondary challenge with B. pertussis. In contrast to the exacerbating effect of FTY720 on a primary infection (Fig. 2A), treatment of mice prior to reinfection did not alter the course of a secondary infection (Fig. 5A). Assessment of CFU in the lungs after secondary challenge with B. pertussis revealed a rapid reduction in bacteria in the lungs by day 2 p.i., as well as complete clearance by day 7 p.i., in untreated and FTY720-treated mice (Fig. 5A). Flow cytometric analysis of cell populations in the lungs revealed that the number of lung CD4 T cells increased rapidly 7 d p.i. in control and FTY720-treated mice. In contrast, the number of circulating CD4 T cells in FTY720-treated and untreated control mice did not increase after reinfection and remained at a low level on day 7 p.i. (Fig. 5B, 5C). The number of circulating CD8 T and B cells increased after reinfection, and these were significantly reduced by treatment with FTY720. In contrast, the number of CD8 T cells and B cells in the lungs on day 7 after reinfection increased, but this was not affected by treatment with FTY720 (Fig. 5B, 5C). Although Ag-specific serum Ab responses were suppressed in mice treated with FTY720 before and during secondary infection (Supplemental Fig. 1), the rapid clearance of bacteria following rechallenge of convalescent mice was not affected by treatment with FTY720 (Fig. 5A). These findings suggest that the CD4 T cells that develop during the primary infection and reside locally in the lungs are likely to mediate the protective immune response that clears a secondary infection.

We next examined Ag-specific T cell responses in convalescent FTY720-treated and untreated mice 1 d before reinfection. Lung CD4 T cells (predominantly T_RM cells) from convalescent mice treated with FTY720 for 9 d produced similar concentrations of IFN-γ and IL-17A as did CD4 T cells from the lungs of untreated mice (Fig. 5D). Similarly, spleen cells from convalescent mice treated with FTY720 for 9 d responded to B. pertussis Ags as efficiently as did spleen cells from untreated mice (Fig. 5D). These results suggest that FTY720 treatment does not affect the function of Ag-specific CD4 T cells, but it does impair migration of lymphocytes during primary infection, which results in a lower number of Ag-specific CD4 T cells in the spleen of mice treated with FTY720 for 70 d (Fig. 4B). The finding that there are similar numbers of B. pertussis–specific CD4 T cells in the lungs of FTY720-treated and untreated mice provides further evidence that lung CD4 cells mediate protection against secondary infection with B. pertussis.

The data presented in Fig. 5 suggest that local CD4 T cells mediate protection against reinfection, whereas the data presented in Fig. 1 suggested that CD4 T_RM cells are expanded significantly after reinfection. Therefore, we characterized T_RM cells in the lungs after reinfection in FTY720-treated and untreated mice. In convalescent mice (60 d postprimary infection), a significant frequency of lung CD4 T cells with an effector memory phenotype (CD44⁺CD62L⁻) express the T_RM cell markers CD69 and CD103 (Fig. 6A). In contrast, expression of CD69 and CD103 was low on circulating CD44⁺CD62L⁻CD4 T cells (Fig. 6A). The frequency (Fig. 6A) and absolute number (Fig. 6B) of lung CD44⁺CD62L⁻ CD4 T cells expressing CD69 and CD103 (T_RM cells) increased after reinfection with B. pertussis. Treatment with FTY720 from day 10 before reinfection did not affect the expansion of CD4 T_RM cells in the lungs, suggesting that these cells proliferate locally in the lungs following reinfection with B. pertussis.

To confirm that CD4 T_RM cells expanded locally in the lungs p.i. and reinfection with B. pertussis, we compared the expression of the proliferation marker Ki67 in lung CD4 T_RM cells and CD4 T_CM cells. T_CM and T_RM cells from the lungs of uninfected mice had minimal expression of Ki67; however, 7 d after primary infection, >50% of T_RM cells expressed Ki67, whereas only ∼10% of T_CM cells proliferated (Fig. 6C). Compared with T_CM cells, lower numbers of T_RM cells were detected in convalescent mice, and neither population expressed Ki67. However, we did detect significant enhancement of Ki67 in T_RM cells, but not in T_CM cells, 7 d after rechallenge of convalescent mice (Fig. 6C). Proliferation of lung T_RM cells, determined by Ki67 staining, was not reduced by treatment of the mice with FTY720 prior to and during reinfection (Fig. 6D), providing convincing evidence of in situ expansion of T_RM cells in the lungs. These findings suggest that CD4 T cells are induced in the lungs during primary infection with B. pertussis, expand locally following reinfection, and help to mediate rapid clearance of bacteria from the respiratory tract.

We have shown that treatment of mice with FTY720 during the course of primary infection with B. pertussis did not affect the accumulation of T_RM cells, but it did reduce the rate of clearance of a primary infection. In contrast, treatment of convalescent animals with FTY720 did not affect T_RM cell expansion in the lungs or the clearance rate of a secondary infection. To evaluate whether lung CD4 T_RM cells mediate clearance of bacteria from the respiratory tract, we adoptively transferred CD4 T cells isolated from either lungs or spleens of convalescent mice and from spleens of naive mice into sublethally irradiated naive animals. Sublethal irradiation selectively depletes lymphocytes and provides an empty T cell compartment, analogous to athymic mice, for cell transfer (5). Mice were challenged 1 d after the transfer, and CFU counts in the lungs were evaluated 13 and 20 d after challenge. The results demonstrate that transfer of splenic and lung CD4 T cells from convalescent animals, but not splenic CD4 T cells from naive mice, significantly reduces bacterial burden in the lungs. Mice that received immune lung CD4 T cells had significantly lower CFU counts in the lungs than did mice that received naive splenic CD4 T cells or PBS (Fig. 7A). Furthermore, on day 13 postchallenge, there were significantly fewer bacteria in the lungs of mice that received lung CD4 T cells from convalescent mice compared with mice that received splenic CD4 T cell from convalescent mice.

We examined CD4 and CD4⁺CD69⁺CD103^+/− T cells in the circulation and the lung (based on CD45 staining) in recipients of the cell transfer 13 d postchallenge. The results revealed that >90% of CD4 T cells in the lungs of mice that received lung T_RM cells or splenic CD4 T cells from convalescent, but not naive, mice were lung-resident, suggesting that the transferred immune CD4 T cells are recruited to the lungs following infection (Fig. 7B). The CD4 T cells that migrated to the lungs had significant expression of CD69 and CD103. There were also greater numbers of total CD4 T cells and CD69⁺ T_RM cells in the lungs of recipient mice 13 d p.i. compared with mice that received naive splenic CD4 T cells or PBS only (Fig. 7C). Collectively, these findings demonstrate that T_RM cells persist in the lungs after clearance of B. pertussis and play a significant role in pathogen clearance from the lungs after secondary infection.

The significant finding of this study is that pathogen-specific CD4 T cells with a T_RM phenotype proliferate locally in the lung following infection and, especially, following reinfection of mice with B. pertussis; furthermore, these cells play a critical role in rapid resolution of a secondary infection with B. pertussis. Our findings are consistent with our previous studies demonstrating that peripheral Th1 and Th17 cells are critical for protective immunity against B. pertussis (4, 8) and reveal that T cells may exert a major part of their effector function against reinfection with B. pertussis locally in the respiratory tissue.

The resurgence of pertussis in the last few years has been linked with the rapid waning of immunity induced following immunization with the current aP vaccine (3). It is generally accepted that immunity induced by previous infection with B. pertussis is more effective than that induced by vaccination. Although B. pertussis–specific memory T and B cells have been documented in the peripheral blood of humans or spleen of mice following infection with B. pertussis or immunization with pertussis vaccines (8, 12), none of these studies examined T cells in respiratory tissue. Our study demonstrates that memory T and B cells that are induced in the LNs migrate into the circulation and can enter the lungs. Consistent with the literature (29), the T_eff cells and T_EM cells predominantly homed to the lung. Despite inhibition of lymphocyte migration, the number of T_EM cells increased in the lungs over the course of infection in FTY720-treated mice, as well as in untreated mice. This may be explained by the fact that tissue-infiltrating T_EM cells can become T_RM cells, which proliferate locally in tissues and play a critical role in immunity to infection (30). Using the combination of an in vivo–labeling technique with anti-CD45 to discriminate circulating (CD45⁺) T cells from lung-resident (CD45⁻) T cells and ex vivo staining for flow cytometry with Abs for memory populations expressing CD103 and CD69, we demonstrated that lung CD4 T cells with a T_EM cell phenotype were predominantly T_RM cells.

Our study suggests that pathogen-specific T cells that are generated in the LNs during infection with B. pertussis play a role in the clearance of a primary infection by differentiating into effector Th1/Th17 cells. Our data, together with published studies (4), suggest that the initial control of a primary infection during the first 2–3 wk is mediated by innate immune responses and then adaptive immune responses, especially effector CD4 T cells, as well as Abs, help to clear the infection over the next 2–3 wk. In the current study, we found that FTY720 treatment prior to and during a primary B. pertussis infection does not affect the bacterial clearance curves over the first 2–3 wk, when the innate response is likely to provide the dominant protective effect; however, it significantly exacerbates the later stages of infection because it prevents migration of the protective CD4 T cells, especially T_eff, T_EM, and T_CM cells, into the lungs. Interestingly, B cell egress from LNs into the circulation and Ab responses in the serum were substantially suppressed in mice treated with FTY720 prior to and during primary infection, suggesting that Abs also contribute to clearance of a primary infection. However, Ab responses were also suppressed, albeit not as dramatically, in mice treated with FTY720 before and during secondary infection, yet the rapid clearance of bacteria following rechallenge of convalescent mice was not affected by treatment with FTY720. During reinfection, T_RM cells are already in the lungs, having developed from infiltrating T cells during the primary infection, and are not affected by FTY720 treatment prior to reinfection; therefore, they may play a major role in the rapid bacterial clearance of the secondary infection.

It has been suggested that T_RM cells may nonspecifically expand in tissues during infection but that Ag-specific T cells migrating from the LNs are required for optimum effector function (30). Our study demonstrated that T_RM cells proliferate in the lungs during infection. Interestingly, we found that the numbers of CD4 T_RM cells in the lung of B. pertussis–infected mice were not affected by treatment with FTY720, whereas naive and T_CM cells were depleted and T_EM cells were partially depleted in the lungs of B. pertussis–infected mice treated with FTY720 prior to and during infection. It is possible that lung T_RM cells specific for unrelated pathogens expand in the lung during infection with B. pertussis, either through recognition of cross-reactive Ags or through nonspecific activation by cytokines. However, CD4 T cells purified from the lungs of infected mice were found to secrete IFN-γ and IL-17 in response to B. pertussis Ags. Furthermore, IFN-γ– and IL-17–producing CD4 T cells were detected by flow cytometry in the lungs of B. pertussis–infected mice. These findings suggest that the expanded T_RM cells were B. pertussis specific and had features of Th1 and Th17 cells (31). The expansion of Th17 cells in the lungs during B. pertussis infection was particularly striking. Interestingly, there was a higher frequency of IL-17– and/or IFN-γ–producing CD4 T cells in the lungs of FTY720-treated mice compared with untreated mice. This can be explained by the fact that T_RM cells are more potent responders to Ag stimulation (32) and that this population dominated in the lungs of mice treated with FTY720.

The most critical function of memory T cells, especially T_RM cells, was observed in protection against reinfection with B. pertussis. We found that these T_RM cells proliferate in the lungs during infection and, in particular, soon after reinfection with B. pertussis. The expansion of CD4 T_RM cells in the lungs following reinfection of convalescent mice was associated with rapid clearance of the bacteria from the respiratory tract. The local expansion of T_RM cells or clearance of the infection was not affected by treatment with FTY720 for 10 d before reinfection; however, circulating CD8 T cells and B cells were significantly reduced in convalescent mice treated with FTY720. This provides further evidence that lung-resident CD4 T_RM cells are reactivated by infection. Moreover, adoptive transfer of CD4 T_RM cells from the lungs of convalescent mice conferred significant protection against B. pertussis challenge to naive irradiated mice. This is consistent with the suggestion that CD4 T_RM cells play an important protective role in the immediate response to infection at mucosal surfaces (15). Our findings suggest that T_RM cells play a crucial role in adaptive immunity induced by previous infection with B. pertussis and may be important targets for effective adaptive immune responses induced by vaccination against pertussis.

We thank Barry Moran for assistance with flow cytometry.

The authors have no financial conflicts of interest.