To test whether a naive immune system has a weaker or stronger immunity than an educated immune system upon new viral infection, we studied the immune system of GF mice without a history of microbial exposure and compared their T-cell responses to LCMV infection with those of Conv mice. Firstly, we sorted CD90.1 P14 CD8 T cells from CD90.1 P14 mice and transferred the same quantity of cells into CD90.2 Conv mice or CD90.2 GF mice, respectively, following acute viral infection with the new pathogen LCMV Armstrong ( Figure 1A ). Then, we analyzed the CD8 T-cell responses on the indicated days. Surprisingly, GF mice exhibited a more robust CD8 T-cell immunity than Conv mice, with both the cell proportion and the cell number of the LCMV-specific P14 CD8 T cells elevated dramatically in the spleen on day 8 ( Figure 1B , Supplementary Table S1 ).

As the microbiota is largely associated with intestinal microbial populations, we also analyzed small intestinal lymphocytes. The intestine contains the largest number of immune cells of any organ in the body, and the intestinal mucosal immune system is composed of three major lymphoid areas: the lamina propria (LP, which lies just underneath the basement membrane in the intestinal villi), the intraepithelial compartment (IEL, which contains intraepithelial lymphocytes and is located between the columnar epithelial cells), and Peyer’s patches (PPs, which are embedded in the gut wall) (12, 13). Here, we first analyzed CD8 T cells in the LP of the small intestine (SI) and found that both the transferred LCMV-specific P14 CD8 T cells and the polyclonal CD44⁺ CD8 T cells of recipient mice were robustly enriched in the GF group compared with those in the Conv group ( Figure 1C ). We also tested CD69⁺CD103⁺ Trm (resident memory T cell) precursors on day 8 in the LP and found that both the cell proportion and the cell number of Trm precursors were overtly increased in GF mice compared with those in Conv mice ( Figure 1D ). We then detected and confirmed that the Trm precursors on day 8 in the IEL also showed a robust increase in both cell proportion and cell number in GF mice compared with those in Conv mice ( Figure 1E , Supplementary Figure S1 ).

Furthermore, to investigate CD4 T-cell responses, we sorted CD45.1 SMARTA CD4 T cells from CD45.1 SMARTA mice and transferred the same quantity of cells into CD45.2 Conv mice or CD45.2 GF mice, followed by infection with LCMV ( Figure 1F ). Then, we analyzed the CD4 T-cell responses on the indicated days. Consistently, both the cell proportion and the cell number of LCMV-specific SMARTA cells were dramatically augmented in GF mice compared with those in Conv mice ( Figure 1G ), followed by obvious increases in both follicular helper T (Tfh) and T helper 1 (Th1) cell numbers ( Figure 1H , Supplementary Figure S3 ). Because of the helper functions of Tfh cells for germinal center B-cell (GC B cell) maturation for antibody production, we further detected both germinal center B cells and plasma cells. Likewise, both the cell proportion and the cell number of GC B cells and plasma cells showed a robust increase in GF mice compared with those in Conv mice ( Figures 1I, J , Supplementary Figure S4 ). These findings demonstrated that mice without a history of microbial education developed robust adaptive immune responses to novel viral infections.

To investigate whether mice without a history of microbial education can establish long-term protection, we first sorted CD90.1/CD45.1 P14 CD8 T cells from CD90.1/CD45.1 P14 mice and transferred the same quantity of cells into CD90.2/CD45.2 Conv mice or CD90.2/CD45.2 GF mice, followed by acute viral infection with the new pathogen LCMV Armstrong ( Figure 2A ). Then, we examined LCMV virus-specific P14 CD8 T cells on day 39 in the spleen and found that both the cell proportion and the cell number of P14 CD8 T cells were robustly increased in GF mice compared with those in Conv mice ( Figure 2B ). We further analyzed CD127⁺KLRG1⁻ memory CD8 T cells, especially CD127⁺CD62L⁺ Tcm (central memory T) cells, in P14 cells on day 75 in the spleen. Consistently, not only memory CD8 T cells but also Tcm cells increased in number in GF mice compared with those in Conv mice ( Figure 2C ). We then examined the polyclonal CD127⁺KLRG1⁻ memory CD8 and CD127⁺CD62L⁺ Tcm cells among CD44⁺ CD8 T cells of the recipient mice. The results were consistent with those for the transferred P14 CD8 T cells, with augmentation of both memory CD8 and Tcm cell levels among the polyclonal CD44⁺ CD8 T cells of the recipient GF mice ( Figure 2D ). As laboratory evidence from clinical patients showed greater clonal expansion of T cells with more IL-2 and IFN-γ-producing T cells in moderate disease than in severe disease for COVID-19, we further applied ICC analysis on day 57 in the spleen, testing for IL-2 and IFN-γ, for a deeper understanding of memory CD8 T-cell functions and found that GF mice showed higher IL-2 and IFN-γ levels in both cell proportion and mean fluorescence intensity (MFI) in both P14 CD8 T cells and polyclonal CD44⁺ CD8 T cells of the recipient GF mice compared with those of Conv mice ( Figures 2E, F , Supplementary Figure S2 , Supplementary Table S1 ).

We also analyzed small intestinal lymphocytes. First, we found that both the cell proportion and the cell number of LCMV-specific P14 CD8 T cells showed a robust increase on day 39 in the IEL of GF mice compared with those of Conv mice ( Figure 2G ). Furthermore, we analyzed CD69⁺CD103⁺ Trm cells among P14 CD8 T cells and found that Trm cells also showed an obvious increase in both cell frequency and cell number on day 39 in the IEL in GF mice compared with those in Conv mice ( Figure 2H ). We then examined both LCMV-specific P14 cells and Trm cells on day 57 in the LP, and the results were consistent with those for the IEL ( Figures 2I, J ). We further confirmed the robust increase in both the cell frequency and the cell number of Trm cells among polyclonal CD44⁺ CD8 T cells of the recipient GF mice ( Figure 2K ). Finally, to verify the phenotype, we also examined P14 cells in another tissue, the lung, with repeatable results showing a robust increase in both the frequency and the number of P14 cells on day 57 in the lungs of GF mice compared with those of conditional mice ( Figure 2L ). Collectively, the above results showed that mice without a history of microbial exposure established more potent memory CD8 T cells for long-term protection against viral reinfection.

Furthermore, we wanted to determine whether naive GF mice have primary lower or higher T-cell levels than Conv mice without LCMV infection. We thus found that both CD8 and CD4 T cells, including regulatory T cells (Tregs with CD4⁺FOXP3⁺), showed a lower primary cell number in naive GF mice than in Conv mice ( Figures 3A–C , Supplementary Table S1 ).

In short, naive GF mice showed lower levels of primary T cells. This finding was consistent with previous reports (13–15). On the other hand, the above overall results indicated greater plasticity with sensitivity of the naive immune system of GF mice, as shown by the robust adaptive immune responses and immune memory established upon new viral infection, even in the condition of primary lower T cell levels for GF mice compared with that of Conv mice.

To investigate whether the microbiota impacts the T-cell responses in Conv mice, we treated mice with antibiotics to eliminate the microbiota ( Figure 4A ). Thus, we compared LCMV-specific P14 CD8 T cells and polyclonal CD44⁺ CD8 T cells of recipients on day 8 in the spleen of the ABX-treated mice with those of normal Conv mice as a control, and we found that both P14 cells and the polyclonal CD8 T cells of the recipient ABX-treated mice showed a decreased cell number ( Figure 4B ). Because of the importance of small intestinal lymphocytes with large intestinal microbiota populations, we also examined P14 cells and polyclonal activated CD8 T cells on day 12 in the IEL and LP, respectively, and found that the results were consistent with those in the spleen; both P14 cells and polyclonal CD8 T cells of the recipient ABX-treated mice exhibited decreased cell numbers in both the IEL and LP ( Figures 4C-F ). Furthermore, we also detected polyclonal memory CD8 T cells and Trm cells for long-term protection on day 61 in the IEL and LP of the recipient ABX-treated mice and Conv mice, with all of them showing a consistently lower number in the ABX-treated mice than in control mice ( Figures 4G–J , Supplementary Table S1 ).

In brief, with the microbiota eliminated, the ABX-treated mice did not show the same immune responses as the GF mice upon new viral infection, compared with normal Conv mice, respectively. The major difference in their immune system is that the ABX-treated mice still have a history of microbial education; in other words, their immune system is not that naive. Although naive GF mice have fewer primary T cells than Conv mice, they exhibited more robust T-cell responses after new viral infection, including an elevated cell proportion and cell function of memory CD8 T cells for long-term protection. The naive immune system of GF mice with a much abundant naive T-cell repertoire seems to have greater plasticity with sensitivity for aspects of T cells, including T-cell activation, proliferation, differentiation, and function, which is consistent in different organs.

P14 (CD90.1 or CD45.1) and SMARTA (CD45.1) TCR transgenic mice were obtained from Dr. Rafi Ahmed (Emory University). C57BL/6J (CD45.2) mice were purchased from the Jackson Laboratories. Germ-free mice were provided by Dr. Hong Wei and the Animal Facility of Third Military Medical University, and these GF mice were bred in sterile plastic isolators with autoclaved food and water. All handling procedures for GF mice, including LCMV infection, were carried out under sterile conditions. All mice analyzed were 6–8 weeks of age, and both male and female mice were included without randomization or blinding. LCMV Armstrong was provided by Dr. Rafi Ahmed, and 2 × 10⁵ PFU (i.v.) were used to establish acute infection in mice. Mice infected with LCMV were housed in accordance with Institutional Biosafety Regulations of Third Military Medical University. All experimental protocols were approved by the Institutional Animal Care and Use Committees of the Third Military Medical University. All methods were carried out in accordance with relevant guidelines and regulations that were approved by the Ethics Committees of the Third Military Medical University. This study was carried out in compliance with the ARRIVE guidelines.

In each individual experiment, a total of 2 × 10⁴ naive CD45.1⁺ (or CD90.1⁺) P14 cells or SMARTA cells were adoptively transferred into naive wild-type CD45.2⁺ (or CD90.2⁺) mice, and then these recipient mice were infected with 2 × 10⁵ PFU LCMV Armstrong (i.v.) the next day.

For the isolation of SI IELs, the small intestine and Peyer’s patches were removed, and the intestine was cut longitudinally into 0.5 cm pieces. The intestine pieces were incubated with 0.154 mg/mL of dithioerythritol (DTE) in 10% HBSS/HEPES bicarbonate (30 min at 37°C, 250 rpm) to extract the IEL. For lamina propria lymphocyte (LPL) isolation, gut pieces were further treated with 100 U/mL of type I collagenase (Worthington, Lakewood, USA) in RPMI 1640, 5% FBS, 2 mM of MgCl₂, and 2 mM of CaCl₂ for 2 h at 37°C, stirring at 250 rpm. For the isolation of lung lymphocytes, the lung was removed and cut into small pieces, and the pieces were incubated with 1.3 mM of EDTA in HBSS (30 min at 37°C, 250 rpm), followed by treatment with 100 U/mL of type I collagenase (Worthington) in RPMI 1640, 5% FBS, 2 mM of MgCl₂, and 2 mM of CaCl₂ (1 h at 37°C, 250 rpm). After enzymatic treatment, lymphocytes were then purified on a 44/67% Percoll gradient (800g at 23°C for 20 min).

Flow cytometry data were acquired by FACSCantoII (BD Biosciences, Franklin Lakes, USA) and analyzed with the FlowJo software (Tree Star, Ashland, USA). The antibodies and reagents used for flow cytometry staining are listed in Supplementary Table S1 . Surface staining was performed in PBS containing 2% BSA or FBS (wt/vol). For CXCR5 staining, cells were stained with purified anti-CXCR5 (BD Biosciences) for 1 h at 4°C, followed by biotinylated antirat immunoglobulin G (IgG) (Jackson ImmunoResearch, West Grove, USA) and then fluorescently labeled streptavidin (eBioscience, San Diego, California) for 30 min on ice. Foxp3 staining was performed with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) after surface staining. For detection of cytokine production, splenocytes were first stimulated with the indicated peptide (0.2 µg/mL), GolgiPlug, GolgiStop, anti-CD107a, and anti-CD107b antibodies (BD Biosciences) at 37°C for 5 h. Following surface staining, intracellular cytokine staining was performed with a Cytofix/Cytoperm Fixation/Permeabilization Kit according to the manufacturer’s instructions.

Mice were provided with autoclaved drinking water supplemented with ampicillin (0.5 mg/mL, Solarbio, Beijing, China), gentamicin (0.5 mg/mL, Solarbio), metronidazole (0.5 mg/mL, Solarbio), neomycin (0.5 mg/mL, Solarbio), and vancomycin (0.25 mg/mL, Solarbio). Antibiotic treatment was started 2 weeks before infection and continued for the duration of the experiments.

Statistical analysis was conducted using Prism 6.0 (GraphPad, San Diego, USA). The unpaired two-tailed t-test with 95% confidence interval was used for the calculation of P-values.