Mucosal-Associated Invariant T Cells Accumulate in the Lungs during Murine Pneumocystis Infection but Are Not Required for Clearance

Pneumocystis is a fungal pathogen that can cause pneumonia in immunosuppressed hosts and subclinical infection in immunocompetent hosts. Mucosal-associated invariant T (MAIT) cells are unconventional lymphocytes with a semi-invariant T-cell receptor that are activated by riboflavin metabolites that are presented by the MHC-1b molecule MR1. Although Pneumocystis can presumably synthesize riboflavin metabolites based on whole-genome studies, the role of MAIT cells in controlling Pneumocystis infection is unknown. We used a co-housing mouse model of Pneumocystis infection, combined with flow cytometry and qPCR, to characterize the response of MAIT cells to infection in C57BL/6 mice, and, using MR1−/− mice, which lack MAIT cells, to examine their role in clearing the infection. MAIT cells accumulated in the lungs of C57BL/6 mice during Pneumocystis infection and remained at increased levels for many weeks after clearance of infection. In MR1−/− mice, Pneumocystis infection was cleared with kinetics similar to C57BL/6 mice. Thus, MAIT cells are not necessary for control of Pneumocystis infection, but the prolonged retention of these cells in the lungs following clearance of infection may allow a more rapid future response to other pathogens.


Introduction
Pneumocystis is a fungus that can cause inapparent pulmonary infection in immunocompetent hosts but can cause potentially life-threatening pneumonia (PCP) in immunocompromised hosts [1]. Patients with HIV infection, as well as those with malignancies, transplants, and autoimmune diseases, are at increased risk of developing PCP [2]. Corticosteroids are a well-defined risk factor for developing PCP in these latter settings, but other drugs, including newer biologics, can also increase this risk.
An improved understanding of host immune responses to Pneumocystis should provide insights into the immune defects that allow PCP to develop and may help to better identify patients at the highest risk of developing PCP. Mucosal-associated invariant T (MAIT) cells are innate T cells that are highly conserved throughout mammalian evolution [3]. MAIT cells are alpha beta T cells that have semi-invariant T-cell receptors, which recognize riboflavin metabolites from pathogens and commensals when presented by major histocompatibility-related molecule 1 (MR1). Of note, MAIT cells develop early in life upon exposure to riboflavin metabolites [4]. Other MR1 ligands include folic acid derivatives and several drug and drug-like molecules [5]. MAIT cells have been associated with several disease categories [6], including infectious diseases, such as HIV/AIDS [7,8], sepsis, and tuberculosis, as well as asthma, type 2 diabetes, and obesity [9]. Following activation, MAIT cells develop effector functions, including secretion of cytokines, primarily IL-17A in mice [10], as well as cytotoxicity [11]. MAIT cells have also been shown to promote tissue repair [4,12]. MAIT cells are increasingly being considered for use in immunotherapy for various cancers and for use in vaccine development [13][14][15][16].
The availability of high-quality Pneumocystis genomes allows us to explore not only the biology of Pneumocystis but also its potential interactions with host immune defenses. The set of genes needed for riboflavin biosynthesis have been identified in all Pneumocystis species except possibly Pneumocystis canis (which appears to be missing a single gene) [17,18], suggesting that MAIT cells could play a role in controlling Pneumocystis infection. To explore this hypothesis, we utilized mouse models of Pneumocystis infection to examine the kinetics of MAIT cells in the lung and whether their absence impacts infection.

Animals
C57BL/6 mice were obtained from the National Institutes of Health (NIH, Bethesda, MD) or The Jackson Laboratory (Bar Harbor, ME). CD40 ligand knockouts (CD40L-KO, strain B6, 129S-Tnfsf5 tm1lmx /J), which are highly susceptible to Pneumocystis infection, were obtained from The Jackson Laboratory. MR1 −/− mice (on a C57BL/6 background), which lack MAIT cells, were generated by Dr. Susan Gilfillan and colleagues [19] and were kindly provided by Drs. Michael Constantinides and Yasmine Belkaid; their genotype was verified by PCR [19]. Mice were subsequently housed in microisolator cages in ventilated racks and bred at the NIH Clinical Center animal facilities. All animal studies were performed under an NIH Clinical Center Animal Care and Use Committee-approved protocol.

P. murina Infection Model
Female mice, typically 6-8 weeks old, were infected by co-housing with female P. murina infected seeder mice (CD40L-KO) and sacrificed at various time points [20,21]. This model more closely resembles natural infection via inhalation of organisms than an intratracheal inoculation model and provides predictable kinetics, with a peak of infection in immunocompetent mice at~5 to 6 weeks and clearance by~9 to 11 weeks post initial exposure. Female mice were utilized because co-housed non-litter males are combative. Unexposed litter mate controls (male and female) were housed in clean facilities; to minimize the number of animals used per experiment, 1-2 unexposed mice per time point were sacrificed and processed simultaneously with exposed mice, and data from these unexposed mice were combined as controls.
To compare infection and immune responses, different strains of mice were co-housed to maintain identical exposure conditions. Blood, lungs, and, in 1 experiment, spleens, from 2 to 4 animals per strain per time point, were collected. Serum was analyzed for anti-P. murina antibodies by ELISA; a portion of the lungs was analyzed for P. murina infection by qPCR; and lung and spleen were processed for flow cytometry.

Quantitation of P. murina by qPCR
To quantitate P. murina organism burden, DNA was extracted from a piece of lung using the QIAamp DNA Mini Kit (Qiagen, Germantown, MD, USA), followed by qPCR targeting the P. murina dihydrofolate reductase gene (dhfr), a single-copy gene, using the ViiA 7 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) or CFX384 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), as previously described [20,22]. Results are expressed as log 10 dhfr copies/mg lung tissue.

ELISA
Anti-P. murina antibodies were measured by ELISA using a crude P. murina lysate as previously described [23]. Briefly, Pneumocystis organisms were partially purified by Ficoll-Hypaque density gradient centrifugation and disrupted with glass beads, followed by sonication and centrifugation; the resulting supernatant was utilized to coat the wells. Wells were incubated with mouse serum (1:100 dilution), followed by secondary antibody (1:1000 dilution; Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA), and developed using OPD substrate (Sigma-Aldrich, Inc., St. Louis, MO, USA).

Flow Cytometry
For single-cell suspensions, lung and spleen tissues were digested with Lung Dissociation Kit, mouse and Spleen Dissociation Kit, mouse (Miltenyi Biotec, Bergisch Gladbach, Germany), respectively, using the gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec), per manufacturer's recommendations.

Flow Cytometry
For single-cell suspensions, lung and spleen tissues were digested with Lung Dissociation Kit, mouse and Spleen Dissociation Kit, mouse (Miltenyi Biotec, Bergisch Gladbach, Germany), respectively, using the gentleMACS Octo Dissociator with Heaters (Miltenyi Biotech), per manufacturer's recommendations.
For flow cytometry analysis, 1-2 million cells per tube were labeled with Live/Dead Fixable Violet Dead Cell Stain (Molecular Probes, Eugene, OR, USA), Fc receptors were blocked, and cells were subsequently labeled with antibodies plus tetramer for 1 h at room temperature and then fixed with fixation buffer (BD Biosciences). Data were acquired on a MACSQuant 10 analyzer or Fortessa LSR Flow Cytometer and analyzed using FlowJo Software (version 10.8.1; BD Life Sciences, Ashland, OR, USA). MAIT cells were present almost exclusively in the CD3 + TCRβ + CD4 − CD8 − (double negative (DN)) T-cell populations and are thus reported as a percent of DN T cells. Figure 1 illustrates the gating strategy for identifying MAIT cells. CD19 + and CD11b + cells were excluded from live lymphocytes singlets. CD3 + TCR − β + T cells were then gated on CD11b − CD19 − cells followed by CD4 and CD8. To identify MAIT cells, CD8 + , CD4 + and CD4 − CD8 − (DN) T cells were analyzed for binding of 5-OP-RU MR1 tetramer. 5-FP MR1 control tetramer was used as a negative control to gate MAIT cells, which were primarily found in the DN T-cell population.

Statistics
Cell populations at individual time points were compared to baseline values using unpaired, two-sided Student's t-test, using Prism (version 9, GraphPad Software, LLC, San Diego, CA, USA) or Excel (version 16.59, Microsoft, Redmond, WA, USA).

MAIT Cells Accumulate in the Lungs of C57BL/6 Mice during P. murina Infection
Given the potential role of MAIT cells in immunity to bacterial and fungal pathogens, we undertook studies to examine their role in mouse models of Pneumocystis infection. To determine whether acute Pneumocystis infection induces accumulation of MAIT cells in the lungs of immunocompetent mice, C57BL/6 mice were euthanized at 2, 3, 5, and 7 weeks following the start of co-housing with an infected seeder, and lungs and spleens were harvested. Low numbers of P. murina organisms were detected by qPCR in the lungs at 2 weeks (mean 310 copies/mg tissue), with a gradual increase over time ( Figure 2A). As previously reported [25], CD4 + T cells as a percent of CD3 + T cells increased in the lungs during infection, while CD8 + T cells decreased ( Figure 2B), and anti-Pneumocystis antibodies developed by week 7 ( Figure 2C). MAIT cells (MR1 5OP-RU tetramer positive) were identified almost exclusively in the DN T-cell population, which accounted for <10% of T cells (Figures 1 and 2B). MAIT cells were present at low frequencies in the lungs of unexposed C57BL/6 mice (~1.7% of DN T cells) but accumulated in the lungs of Pneumocystis-infected mice after 2 weeks (mean, 2.9% of DN T cells), peaking at 7 weeks' (9.7% DN T cells) exposure ( Figure 2D). The trends were similar when MAIT cells were characterized as a percent of total CD3 + T cells rather than DN T cells. During this same time frame, MAIT cells accounted for <1.0% of DN T cells in the spleen through week 5, with a small increase to 1.3% at week 7 (data not shown).
control tetramer was used as a negative control to gate MAIT cells, which were primarily found in the DN T-cell population.

Statistics
Cell populations at individual time points were compared to baseline values using unpaired, two-sided Student's t-test, using Prism (version 9, GraphPad Software, LLC, San Diego, CA, USA) or Excel (version 16.59, Microsoft, Redmond, WA, USA).

MAIT Cells Accumulate in the Lungs of C57BL/6 Mice during P. murina Infection
Given the potential role of MAIT cells in immunity to bacterial and fungal pathogens, we undertook studies to examine their role in mouse models of Pneumocystis infection. To determine whether acute Pneumocystis infection induces accumulation of MAIT cells in the lungs of immunocompetent mice, C57BL/6 mice were euthanized at 2, 3, 5, and 7 weeks following the start of co-housing with an infected seeder, and lungs and spleens were harvested. Low numbers of P. murina organisms were detected by qPCR in the lungs at 2 weeks (mean 310 copies/mg tissue), with a gradual increase over time ( Figure 2A). As previously reported [25], CD4 + T cells as a percent of CD3 + T cells increased in the lungs during infection, while CD8 + T cells decreased (Figure 2B), and anti-Pneumocystis antibodies developed by week 7 ( Figure 2C). MAIT cells (MR1 5OP-RU tetramer positive) were identified almost exclusively in the DN T-cell population, which accounted for <10% of T cells (Figures 1 and 2B). MAIT cells were present at low frequencies in the lungs of unexposed C57BL/6 mice (~1.7% of DN T cells) but accumulated in the lungs of Pneumocystisinfected mice after 2 weeks (mean, 2.9% of DN T cells), peaking at 7 weeks' (9.7% DN T cells) exposure ( Figure 2D). The trends were similar when MAIT cells were characterized as a percent of total CD3 + T cells rather than DN T cells. During this same time frame, MAIT cells accounted for <1.0% of DN T cells in the spleen through week 5, with a small increase to 1.3% at week 7 (data not shown).  For all panels, for exposed mice n = 3 each for weeks 2, 3, and 5, and n = 2 for week 7, and for unexposed controls, n = 4.

MAIT Cells Are Not Required for Control of P. murina Infection
Since MAIT cells accumulated in the lung of C57BL/6 mice during Pneumocystis infection, we next sought to determine whether MAIT cells impact clearance of infection and whether the absence of MAIT cells would affect anti-P. murina antibody responses. To address this question, we co-housed MR1 −/− mice, which lack MAIT cells, and C57BL/6 mice with P. murina infected seeder mice. We found that after 5 weeks, MR1 −/− mice had similar P. murina organism loads as C57BL/6 mice and that both groups had cleared infection by week 11, demonstrating that MAIT cells are not required to control or clear P. murina infection ( Figure 3A). Similar levels of anti-P. murina antibodies were present in serum by week 5 in both MR1 −/− and C57BL/6 mice (mean O.D. 0.29 for both) with increases by week 11 (mean O.D. MR1 KO 1.14 and WT 0.90) ( Figure 3B). MAIT cells were detected by flow cytometry at week 5 in C57BL/6 mice but not in MR1 −/− mice, verifying the phenotype of the latter strain ( Figure 3C). bodies using a crude antigen preparation of partially purified P. murina organisms. (D) Flow cyt etry results demonstrating MAIT cells detected in the lungs of C57BL/6 mice using 5-OP-RU loa MR1 tetramer, shown as percent of CD4 − CD8 − (DN) T cells. Data points represent the mean valu each time point, error bars represent standard deviations and p-values < 0.05 when compare baseline values in panels (B,D) are indicated by *. For panel (B) the color of the asterisks indic which cell population it refers to. For all panels, for exposed mice n = 3 each for weeks 2, 3, an and n = 2 for week 7, and for unexposed controls, n = 4.

MAIT Cells Are Not Required for Control of P. murina Infection
Since MAIT cells accumulated in the lung of C57BL/6 mice during Pneumocystis fection, we next sought to determine whether MAIT cells impact clearance of infect and whether the absence of MAIT cells would affect anti-P. murina antibody respon To address this question, we co-housed MR1 −/− mice, which lack MAIT cells, and C57B mice with P. murina infected seeder mice. We found that after 5 weeks, MR1 −/− mice h similar P. murina organism loads as C57BL/6 mice and that both groups had cleared in tion by week 11, demonstrating that MAIT cells are not required to control or clea murina infection ( Figure 3A). Similar levels of anti-P. murina antibodies were presen serum by week 5 in both MR1 −/− and C57BL/6 mice (mean O.D. 0.29 for both) with creases by week 11 (mean O.D. MR1 KO 1.14 and WT 0.90) ( Figure 3B). MAIT cells w detected by flow cytometry at week 5 in C57BL/6 mice but not in MR1 −/− mice, verify the phenotype of the latter strain ( Figure 3C). For both panels, for exposed mice n = 3 each for weeks 5 and 11, and n = 4 for week 23 for each strain, and for unexposed controls, n = 3 for MR1 −/− and n = 6 for C57BL/6 mice. All mice were genotyped to confirm they were the correct strain.

MAIT Cells Remain Elevated in Lungs after Clearance of P. murina Infection
Given that MAIT cells may play a role in protecting against other respiratory pathogens, including bacteria and viruses, such as influenza, we wanted to determine whether MAIT cells remained in the lungs of mice for a prolonged period of time following clearance of P. murina infection. Immunocompetent C57BL/6 mice typically clear infection by week 9-11 post-exposure. To determine whether MAIT cells were still increased in the lungs following clearance, we looked at MAIT cells in the lungs of C57BL/6 mice for up to 22 weeks after initial exposure. C57BL/6 mice were infected by week 5 (mean 1986 copies/mg tissue) and had cleared infection by week 9, as expected; changes in lymphocyte populations and anti-P. murina antibodies were consistent with prior studies (Figure 4A-C) [25]. MAIT cells were elevated above controls from weeks 9 to 22, though only statistically significantly elevated at week 16 (5.7% of DN T cells, Figure 4D). When expressed as a percent of CD3+ T cells, MAIT cells were above control values at weeks 16 and 22, though neither was statistically significant. Similarly, in mice that were exposed to a seeder for only 1 week rather than continuously, MAIT cells accounted for 6.3% of DN T cells at week 20.
data points represent the mean value at each time point, and error bars represent standard d tions. MR1 −/− and C57BL/6 mice showed no significant difference in organism burden, kinetics/ ance of infection or development of anti-Pneumocystis antibodies. For both panels, for exposed n = 3 each for weeks 5 and 11, and n = 4 for week 23 for each strain, and for unexposed contro 3 for MR1 −/− and n = 6 for C57BL/6 mice. All mice were genotyped to confirm they were the c strain.

MAIT Cells Remain Elevated in Lungs after Clearance of P. murina Infection
Given that MAIT cells may play a role in protecting against other respir pathogens, including bacteria and viruses, such as influenza, we wanted to deter whether MAIT cells remained in the lungs of mice for a prolonged period of time fo ing clearance of P. murina infection. Immunocompetent C57BL/6 mice typically cle fection by week 9-11 post-exposure. To determine whether MAIT cells were still incre in the lungs following clearance, we looked at MAIT cells in the lungs of C57BL/6 for up to 22 weeks after initial exposure. C57BL/6 mice were infected by week 5 (m 1986 copies/mg tissue) and had cleared infection by week 9, as expected; changes in phocyte populations and anti-P. murina antibodies were consistent with prior studies ure 4A-C) [25]. MAIT cells were elevated above controls from weeks 9 to 22, though statistically significantly elevated at week 16 (5.7% of DN T cells, Figure 4D). Whe pressed as a percent of CD3+ T cells, MAIT cells were above control values at wee and 22, though neither was statistically significant. Similarly, in mice that were exp to a seeder for only 1 week rather than continuously, MAIT cells accounted for 6.3 DN T cells at week 20.

Discussion
In the current study, we showed that MAIT cells, which recognize riboflavin metabolites, accumulate in the lungs of immunocompetent C57BL/6 mice during Pneumocystis infection, but the absence of MAIT cells does not delay or prevent clearance. Moreover, MAIT cells are retained in the lungs following Pneumocystis infection well beyond the time that organisms can be detected.
MAIT cells require riboflavin (vitamin B2) metabolites for development and are primarily activated when these metabolites are presented to their semi-invariant T-cell receptor by MR1, an MHC class 1b molecule; they can also be activated through TLRs and directly by cytokines [3,4]. The influx of MAIT cells into the lungs of immunocompetent mice infected with Pneumocystis provides support for the inference from genome sequencing studies that most Pneumocystis species have the necessary enzymes to synthesize riboflavin [17,18], although it remains possible that they are activated by an alternative mechanism, as noted above.
While Pneumocystis infection leads to an accumulation of MAIT cells in the lung, the similar kinetics of infection and antibody responses in C57BL/6 and MR1 −/− mice clearly demonstrate that MAIT cells are not needed for control of infection. However, it is possible that MAIT cells play a minor role in altering the kinetics of infection; we did not conduct more detailed kinetics, as our primary goal was to determine if Pneumocystis infection could be cleared in the absence of MAIT cells.
In mice, activated MAIT cells primarily produce IL-17A, a cytokine important for control of some fungal infections, but not Pneumocystis, as IL-17A knockout mice can control the infection similarly to C57BL/6 mice [26]. We attempted to characterize the cytokine profile of lung MAIT cells during infection. While IL-17 seemed to be the main cytokine produced, the loss of a high proportion of MAIT cells following in vitro activation, possibly due to MAIT cell death or downregulation of the T-cell receptor, did not allow firm conclusions in this regard.
We also found that CD40 KO and CD40L KO mice, both of which are highly susceptible to Pneumocystis infection, had measurable levels of MAIT cells similar to those found in C57BL/6 mice (data not shown), suggesting that MAIT cells are insufficient to control infection in the setting of a defective adaptive immune response.
MAIT cells were retained in the lungs for many weeks following clearance of Pneumocystis, similar to what has been reported for other infections, such as Salmonella typhimurium [10], where MAIT cells accumulated rapidly following intranasal inoculation and remained elevated through 7 weeks. Since humans and other host species are exposed to Pneumocystis at a very young age, this retention of MAIT cells following infection may provide a benefit to the host, as MAIT cells would be on site to respond more rapidly to other infections in which they may play a more important role.
It is important to note that MAIT cells are much more abundant in humans than mice [27], and thus, the conclusions of this study may not be applicable to humans. In transgenic models with larger numbers of MAIT cells, the importance of these cells in controlling infection with, e.g., E. coli, has been easier to demonstrate [28]. However, MAIT cells are much less numerous in infants under 2 years of age compared to older humans [27], and nearly all humans appear to be infected with Pneumocystis prior to age 1 [29], suggesting that at least for primary infection, the results may be applicable to humans.
We did not have lung weights available and thus could not express changes in MAIT cells per mg lung tissue; it is possible that these changes represent relative and not absolute increases in lung MAIT cells. However, we noted an increase in the percent of CD3 + T cells in co-housed mice compared to controls (data not shown), and we have previously shown by immunohistochemistry that there was an increase in lymphocytes, primarily CD4 + T cells and B cells, as well as macrophages, in C57BL/6 mice infected with Pneumocystis [25]. When MAIT cells were expressed as a percent of CD3 + T cells, similar trends were seen as when they were expressed as a percent of DN T cells.
In summary, MAIT cells accumulate in the lungs in response to murine Pneumocystis infection but do not appear to have a role in controlling or clearing infection. MAIT cells are retained in the lungs for many weeks after infection, which may have a beneficial role in controlling future pulmonary infections.

Data Availability Statement:
The data presented in this study are available within the article.