Intranasal Vaccination with rePcrV Protects against Pseudomonas aeruginosa and Generates Lung Tissue-Resident Memory T Cells

Tissue-resident memory T (TRM) cells are immune sentinels that bear a key role in the local immune system and rapidly respond to infection. Our previous studies showed that mucosal immunization via intranasal pathways was more effective than intramuscular route. However, the mechanism of enhanced protective immunity remains unclear. Here, we formulated a Pseudomonas aeruginosa vaccine composed of type III secretion protein PcrV from P. aeruginosa and curdlan adjuvant and then administered by the intranasal route. Flow cytometry and immunofluorescence staining showed that the ratio of CD44+CD62L−CD69+CD4+ TRM cells induced by this vaccine was significantly increased, and IL-17A production was notably enhanced. Further analysis revealed that vaccinated mice can protect against the P. aeruginosa challenge even after administration with FTY720 treatment. What is more, our results showed that CD4+ TRM might be involved in the recruitment of neutrophils and provided partial protection against Pseudomonas aeruginosa. Taken together, these data demonstrated that CD4+ TRM cells were elicited in lung tissues after immunization with rePcrV and contributed to protective immunity. Furthermore, it provided novel strategies for the development of vaccines for P. aeruginosa and other respiratory-targeted vaccines.

aeruginosa infections [12], because of its high diversity and variability.
Tissue-resident memory (T RM ) cells are a new subpopulation of memory T cells recently identified, which embedded within peripheral tissues [13][14][15]. T RM cells serve as immune sentinels at the respiratory tract and provide rapid and broad-spectrum protective effects against a variety of respiratory infection pathogens [15][16][17]. Induction of memory T and B cells has now been widely accepted as the principal disciplines for effective vaccine design which could provide robust protective immunity against pathogens caused by prior infection [18][19][20]. Both CD4 and CD8 T RM reside in mucosal could be produced by natural infection [21]; however, natural infection could be lethal. Thus, finding an effective way to induce highly protective T RM cells could be an ideal choice especially for the prevention of P. aeruginosa. Previous study showed the type of vaccines and adjuvants, and the route of vaccination could influence the efficacy of T RM . For pulmonary infectious diseases, mucosal immunization via the intranasal pathways is more effective than intramuscular route in inducing and stimulating immune protection of T RM [20,22]. Th17 has been regarded as a major player in the anti-P. aeruginosa immunity; indeed, in our previous study, we identified a soluble P. aeruginosa antigen called rePcrV which could induce Th17 response and provide protection against P. aeruginosa by intranasal immunization [23]. Another substrate, 1,3-β-glucan, derived from Alcaligenes faecalis, has also been reported to prompt a Th1/Th17 response [24,25]. Therefore, we combined rePcrV and 1,3β-glucan supplemented with curdlan as an adjuvant. After immunization with the vaccine by intranasal administration, we observed that the ratio of CD44 + CD62L -CD69 + CD4 + T RM cells induced by this vaccine was significantly increased, and IL-17A production of this subpopulation was notably enhanced after in vitro stimulation. Vaccinated mice infected with P. aeruginosa showed a sharp reduction in the bacterial burden. What is more, our results showed that CD4 + T RM may involve the recruitment of neutrophils and provide partial protection against P. aeruginosa. Better understanding the underline mechanism could provide new strategies for the development of vaccines for P. aeruginosa and other respiratory-targeted vaccines.

Statistical
Analysis. Data are presented as mean ± SEM. Student's t-test and Mann-Whitney U test were conducted, according to the data distribution. The survival rate was analyzed by the Kaplan-Meier survival curves. GraphPad Prism 8.0 (GraphPad Software) was used for data analyses. P values less than 0.05 were considered significant.

CD4 T Cells Were Essential for rePcrV-Mediated
Protection in P. aeruginosa Pneumonia. To inquire the role of lymphocyte-mediated immune responses during rePcrVinduced protection, adult female CB-17 SCID mice were vaccinated with rePcrV plus curdlan or rePcrV plus aluminum. Mice were challenged with P. aeruginosa XN-1 and were observed to survive for 14 days. As shown in Figure 2(a), there was no statistical difference (P = 0:1316) in survival rate between rePcrV-immunized SCID mice and -unimmunized mice, indicating that a complete lymphocyte system was required for protection after rePcrV immunization in P. aeruginosa pneumonia. In order to determine the relative requirements for humoral immunity and cellular immunity, rePcrV vaccine tested the protection in μMT mice (which lack mature B cells), CD8 T cell KO mice, and CD4-depleted mice (by intraperitoneal injection of anti-CD4 antibody GK1.5). As shown in Figure 2(b), rePcrV-immunized μMT mice were significantly protected (P < 0:001) after P. aeruginosa XN-1 challenge, compared with unimmunized mice which were not protected. The result of CD8 T cell KO mice was the same (P < 0:001, Figure 2(c)). However, the rePcrV-immunized CD4depleted mice (P = 0:4728, Figure 2(d)) were not protected after P. aeruginosa XN-1 challenge. These data suggested the key role for CD4 T cells in mediating protection after immunization with rePcrV.

Intranasal Vaccination with rePcrV
Initiates the CD4 + T RM Cell Response. The result above showed that CD4 + T cells are essential for the anti-P. aeruginosa immunity. However, it is still unknown whether circulating or resident CD4 + T cell is the major player. To this end, the lungs were dissociated into a single cell suspension and detected by flow cytometry. A dramatic increase in CD4 + CD44 +-CD62L -CD69 + T RM cells was observed in vaccinated mice compared with unimmunized mice (P < 0:001, Figure 3(a)). Transcriptional analysis of T RM cells showed that they expressed a unique transcription factor profile. Since Hobit together with Blimp-1 regulates the differentiation and maintenance of T RM cells [36], we purified T RM cells from immunized or unimmunized mice and determined the level of Hobit, Blimp-1, RORγt, and T-bet mRNA. As shown in Figure 3(b), the level of Hobit, Blimp-1, and RORγt was increased in mice immunized with rePcrV compared with unimmunized (P < 0:001, respectively). To examine the expression of IL-17A production in CD4 + T RM cells, we employed immunofluorescence staining. The result revealed that the IL-17A expression was enhanced in immunized mice (Figures 3(a) and 3(c)). Representative gating strategies were shown in figure S1.

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Journal of Immunology Research lymphoid organs to the tissue). We found that FTY720 treatment followed by a P. aeruginosa XN-1 challenge induced higher survival in immunized mice (P < 0:0001) but not in unimmunized mice (Figure 4(a)). Vaccine efficacy was maintained in vaccinated mice with FTY720 treatment, as measured indirectly by global disease score (Figure 4(b)) and weight loss (Figure 4(c)). Furthermore, the bacterial load of immunized mice treated with FTY720 decreased significantly (P < 0:01, Figure 4(d)). In contrast, immunized mice significantly alleviated pathological damage (P < 0:001 , Figure 4(e)). It should be noted that, compared with immunized mice without FTY720 treatment, immunized mice with FTY720 treatment diminished partial protection, which suggested that circulating T cells also played a role in preventing P. aeruginosa infection.

rePcrV Vaccine Efficacy
Depended on IL-17A Expression by CD4 + T RM Cells and Remained Independent of IL-7. Lung CD4 + T RM cells in vaccinated mice with FTY720 treatment showed higher level of IL-17A secretion compared with cells from FTY720-treated unimmunized mice (P < 0:01, Figures 5(a) and 5(b)). We treated mice with anti-IL-17A antibody and anti-IFN-γ antibody before and during vacci-nation to determine whether IL-17A or IFN-γ was required for rePcrV vaccine efficacy in the lungs. Anti-IL-17A-immunized mice were not protected against the challenge of P. aeruginosa XN-1 ( Figure 5(c)). In line with this, the histological analysis of the lung tissues of anti-IL-17A-immunized mice revealed a further increase of peribronchial inflammatory cell infiltration (P < 0:001, Figure 5(d)). IL-7 signaling is regarded as a key mediator for homeostatic proliferation of CD4 T cells, which could explain the long-term and circulatory independent maintenance of T RM cells. To assess whether IL-7 mediated the population expansion of T RM cells and contributed to its survival, we applied a neutralizing antibody to IL-7 (anti-IL-7) at days 27, 30, 32, and 34 of the first immunization (day 0). The results showed that neutralization of IL-7 did not increase the bacterial load (P = 0:7836, Figure 6(a)), and there was no statistical difference in histopathological examination between groups (P = 0:1599, Figure 6(b)).
3.6. Depletion of Neutrophils Impaired the Clearance of Pseudomonas aeruginosa from the Lung. Neutrophils are main orchestrators of lung inflammation and play a unique role in the connection between innate and adaptive  Figure 6: rePcrV vaccine efficacy remains independent of IL-7. (a) Experimental timeline. Lung CFU 24 hours after infection in naïve mice, rePcrV-immunized mice, and FTY720 treatment-immunized mice with or without anti-IL-7 Ab (n = 4). (b) Representative H and E stains in naïve mice, rePcrV-immunized mice, and FTY720 treatment-immunized mice with or without anti-IL-7 Ab (n = 5). Significant differences were calculated using unpaired t-test. The "n.s." means "no significant difference." 11 Journal of Immunology Research immunity [39]. In order to investigate whether neutrophils play a role in CD4 + T RM cells mediated protection against P. aeruginosa, neutrophils were deleted before challenge. The results showed that in the neutrophil depletion mice, CFU counts were increased in the lungs of mice treated with anti-Ly6G (P < 0:01, Figure 7(a)), and lung damage was worse (P < 0:0001, Figure 7(b)). Flow cytometry showed that neutrophil depletion did not impact the CD4 + T RM cell population (P = 0:7296, Figure S2). These data indicated that CD4 + T RM may be involved in recruitment of neutrophils and provided partial protection against P. aeruginosa.

Discussion
According to the different types of cytokines secreted, CD4 + T RM cells are divided into Th1, Th2, or Th17 subtypes. Generally, CD4 + T RM cells in viral infection and tumors mainly secreted IFN-γ, while CD4 + T RM cells induced by bacterial or fungal infection mainly expressed IL-17A. The study indi-cated that dermal Candida albicans infection preferentially produces CD4 + IL-17A + T RM cells. When reinfected with Candida albicans, T RM cells could rapidly clear infection challenges [40]. Previous work showed that lung T RM cells were elicited by heat-killed K. pneumoniae [41]. By using IL-17A tracking-fate mouse models [42], CD4 + T RM cells were found derived from effector Th17 cells [27]. Our previous study found that rePcrV could induce Th17 response and enhanced protection [23]. The results of this study initially demonstrate that rePcrV intranasal immunization could induce the generation of CD4 + T RM cells secreting IL-17A in lung tissues of mice, and these cells produced a protective immune response after P. aeruginosa infection. Therefore, the origin of CD4 + IL-17A + T RM cells and their relationship with Th17 cells need to be further investigated in subsequent experiments.
FTY720 not only blocks the egress of T cells but also prevents migration of B cells from lymph nodes to the circulation [15,43]. Indeed, FTY720 treatment appeared to affect  hours after infection in FTY720 treatment mice with or without anti-Ly6G Ab and FTY720 treatment-immunized mice with or without anti-Ly6G Ab (n = 7). (b) Representative H and E stains in FTY720 treatment mice with or without anti-Ly6G Ab and FTY720 treatment-immunized mice with or without anti-Ly6G Ab (n = 7). Significant differences were calculated by using Student's t-test. * P < 0:05; * * P < 0:01; * * * P < 0:001; * * * * P < 0:0001. bacterial burdens and survival in the immunized group, suggesting that circulating T cells or antibody-producing cells were also required in preventing P. aeruginosa infection. However, treatment with FTY720 did not affect T RM cell expansion in the lungs. Our data showed that there was no statistical significance between mice with FTY720 treatment and mice without FTY720 treatment ( Figure S3).
Long-term survival in peripheral tissues is another important characteristic of T RM cells [13,14,44]. Furthermore, researches showed that the survival and expansion of T RM cells in peripheral tissues were mainly regulated by the local immune microenvironment. The formation of the local microenvironment was associated with the involvement of multiple cytokines, such as IL-2, IL-7, IL-15, and TGF-β [45][46][47]. It also showed that multiple correlated signaling pathways may be involved in the maintenance induction of T RM cells in peripheral tissues, including PI3K/Akt, JAK/STAT5, and Notch signal pathways [48]. In our research, we found that neutralization of IL-7 did not affect rePcrV vaccine efficacy, and there was no significance in the bacterial load ( Figure 6). Regrettably, our work did not yet clarify the mechanisms of T RM cell survival and amplification. We will continue to explore them in the future.
Studies have reported that the acellular pertussis vaccine vaccinated with intramuscular injection has a relatively short immunoprotection period and has no obvious effect on the colonization and transmission of B. pertussis in the nasal cavity [49]. On the contrary, nasal inoculation of attenuated pertussis vaccine BPZE1 can resist the infection caused B. pertussis [50]. Comparing the intranasal or injected influenza vaccines, we found that the route of administration and the type of vaccines (inactivated vaccine and live vaccine) also affect the production of CD4 + T RM cells. Nasal vaccination with a live attenuated influenza vaccine (Flu-Mist) induced antigen-specific CD4 + T RM cells in the lung, mediating long-term protection against heterologous influenza virus strains. However, inactivated influenza virus vaccine (Fluzone) did not elicit T RM cell production after nasal inoculation but induced strain-specific neutralizing antibody production [50]. Hence, choosing the appropriate vaccination route and vaccine type is an important means to induce respiratory T RM cell production.

Data Availability
All the data are included in this paper.

Ethical Approval
The animal study was reviewed and approved by the Animal Ethical and Experimental Committee of the Army Medical University. cstc2020jcyj-msxmX0296, 2022NSCQ-MSX4822, and 2021XZL02). Figure S1: representative gating strategies for CD44 +-CD62L -CD69 + CD4 + T RM cells in the lungs. CD44 +-CD62L -CD69 + CD4 + T RM cells were gated on live CD45cells. Figure S2: the graph indicates the number of CD4 +-CD44 + CD69 + CD62L -T cells per mouse (n = 4) found in the lungs of FTY720 treatment-immunized mice with or without anti-Ly6G Ab. Figure