B cell deficiency induces cytotoxic memory CD8+ T cells during influenza-associated bacterial pneumonia

Abstract

Influenza-associated bacterial superinfections in the lung lead to increased morbidity and mortality. Nearly all people have preexisting memory to influenza virus, which can protect against subsequent infection in the lung. This study explored the role B cells play in protection against bacterial (Staphylococcus aureus or Klebsiella pneumoniae) superinfection with previous heterotypic influenza memory. B cell deficiency resulted in an increased inflammatory lung environment and lung tissue injury during superinfection. Loss of B cells increased populations of memory CD8+ T cells in the lung, and these CD8+ T cells were transcriptionally and functionally distinct from those of WT mice. Use of antibody-deficient mouse models showed that this phenotype was specifically due to loss of antibody production from B cells. Passive immunization with influenza antibody serum in B cell-deficient mice rescued the CD8+ T cell phenotype. CD8+ T cell depletion and lethal superinfection challenge experiments showed that the cytotoxic memory CD8+ T cells from B cell-deficient mice protect against superinfection bacterial burden and mortality. These findings provide insight into the importance of B cells for regulating immune responses against infection.

Keywords: Adaptive immunity; Immunology; Infectious disease; Influenza; T cells.

Figure 1. Loss of B cells increases lung populations of T cells and inflammatory macrophages during memory superinfection.

(A) Infection scheme for memory-superinfected mice. IT, intratracheal. (B) Representative H&E lung images (original magnification, ×40) with a heatmap overlay to signify lung areas with inflammation/consolidation. (C) Lung nucleated cell density (cells/mm²) and frequency of lung inflammation and consolidation detections between WT and μMT lungs and between F/F and F/F/S treatment groups (WT-F/F: n = 12; WT-F/F/S: n = 18; μMT-F/F: n = 12; μMT-F/F/S: n = 18). (D) Flow cytometry analysis was conducted on WT and μMT mice with F/F and F/F/S infections. Samples were concatenated (n = 4), and populations were visualized by FlowSOM and t-SNE–CUDA using Cytobank software. (E and F) Samples were analyzed by flow cytometry. Absolute number of CD64⁺CD11b⁺CD11c^lo cells (WT-F/F: n = 12; WT-F/F/S: n = 11; μMT-F/F: n = 12; μMT-F/F/S: n = 13) and percentage iNOS⁺MHCII^hi cells of CD64⁺ cells (WT-F/F: n = 7; WT-F/F/S: n = 8; μMT-F/F: n = 7; μMT-F/F/S: n = 8). Data are represented as mean ± SEM, and P values were determined by repeated 1-way ANOVA measures (**P < 0.01, ***P <0.001, ****P < 0.0001).

Figure 2. Loss of B cells alters CD8⁺ T cell number and phenotype during memory superinfection.

Flow cytometry analysis was conducted on WT and μMT mice with F/F and F/F/S infections. (A and B) Percentage (WT-F/F: n = 8; WT-F/F/S: n = 20; μMT-F/F: n = 8; μMT-F/F/S: n = 24) and absolute number (WT-F/F: n = 8; WT-F/F/S: n = 7; μMT-F/F: n = 8; μMT-F/F/S: n = 8) of CD90.2⁺CD8⁺ cells. (C) Percentage of CD4⁺CD90.2⁺ cells (WT-F/F: n = 8; WT-F/F/S: n = 7; μMT-F/F: n = 8; μMT-F/F/S: n = 8). (D) Absolute number of CD8⁺CD69⁺CD103^hi cells (WT-F/F: n = 8; WT-F/F/S: n = 7; μMT-F/F: n = 8; μMT-F/F/S: n = 8). (E) Intracellular flow cytometry plot (left) showing the percentage granzyme B⁺CD8⁺ T cells using concatenated F/F/S infected WT and μMT samples (WT-F/F/S: n = 4; μMT-F/F/S: n = 4) and absolute number of granzyme B⁺CD8⁺ T cells (right) (WT-F/F: n = 8; WT-F/F/S: n = 7; μMT-F/F: n = 8; μMT-F/F/S: n = 8). (F–H) Percentage of IFN-γ⁺CD8⁺ cells (WT-F/F: n = 12; WT-F/F/S: n = 11; μMT-F/F: n = 12; μMT-F/F/S: n = 11), TNF-α⁺CD8⁺ cells, and Tbet⁺CD8⁺ cells (WT-F/F: n = 8; WT-F/F/S: n = 7; μMT-F/F: n = 8; μMT-F/F/S: n = 8). Data are represented as mean ± SEM, and P values were determined by repeated 1-way ANOVA measures (*P < 0.05, **P < 0.01, ***P <0.001, ****P < 0.0001).

Figure 3. Loss of B cells alters the transcriptional state of lung CD8⁺ T cells during memory superinfection.

Bulk RNA sequencing was performed on lung CD8⁺ T cells from WT and μMT mice with either F/F or F/F/S infections (WT-F/F: n = 4; WT-F/F/S: n = 4; μMT-F/F: n = 3; μMT-F/F/S: n = 3). (A) Venn diagram shows the number of statistically significant differentially expressed genes (DEGs) shared between WT-F/F and WT-F/F/S groups and between μMT-F/F and μMT-F/F/S groups. (B) Top and bottom 10 Gene Ontology pathways in μMT-F/F/S versus WT-F/F/S for genes with false discovery rate adjusted P values less than 0.05 (8,831 DEGs). (C) Clustering heatmap of log₂-transformed transcripts per million (TPM) values of WT-F/F/S and μMT-F/F/S mice for CD8⁺ T cell genes with adjusted P values (right). (D) Flow cytometry analysis showing the percentage of PD-1⁺LAG-3⁺TIM-3⁺CD8⁺ T cells (WT-F/F: n = 11; WT-F/F/S: n = 10; μMT-F/F: n = 11; μMT-F/F/S: n = 12). (E) Gene set enrichment analysis (GSEA) of a pathway in μMT-F/F/S versus WT-F/F/S (top) with P value and enrichment score (bottom). Right: Flow cytometry analysis showing the percentage of annexin V⁺7-AAD^–CD8⁺ T cells (WT-F/F: n = 8; WT-F/F/S: n = 7; μMT-F/F: n = 7; μMT-F/F/S: n = 8). Graphed data are represented as mean ± SEM, and P values were determined by repeated 1-way ANOVA measures (**P < 0.01, ***P <0.001, ****P < 0.0001).

Figure 4. Loss of influenza virus–specific antibody drives cytotoxic memory CD8⁺ T cell responses in superinfection.

(A) X-31 influenza virus–specific serum titers measured by hemagglutinin inhibition assay (HAI) at day 61 (WT: n = 19; MD4: n = 5; μMT: n = 8; IgMi: n = 8). (B) Percentage weight loss was calculated starting from PR8 infection (WT: n = 18; MD4: n = 14; μMT: n = 26; IgMi: n = 7). (C) Viral burden (PR8) assessed via quantitative PCR (WT: n = 19; MD4: n = 14; μMT: n = 16; IgMi: n = 8). (D) Number of MRSA colonies from lung homogenates (WT: n = 22; MD4: n = 9; μMT: n = 18; IgMi: n = 8). (E) Percentage of CD8⁺ cells (WT: n = 27; MD4: n = 11; μMT: n = 27; IgMi: n = 8). (F) Percentage of CD44^hiCD62^loCD8⁺ cells (WT: n = 24; MD4: n = 11; μMT: n = 16; IgMi: n = 8). (G) Absolute number of NP-tetramer⁺CD44^hiCD8⁺ cells (WT: n = 19; MD4: n = 11; μMT: n = 16; IgMi: n = 8). (H) Percentage of PD-1⁺LAG-3⁺TIM-3⁺CD8⁺ cells (WT: n = 24; MD4: n = 11; μMT: n = 16; IgMi: n = 8). (I) Percentage of IFN-γ⁺CD8⁺ cells (WT: n = 19; MD4: n = 7; μMT: n = 20; IgMi: n = 7). (J) Median fluorescence intensity (MFI) of granzyme B⁺CD8⁺ T cells (WT: n = 14; MD4: n = 11; μMT: n = 13; IgMi: n = 7). (K and L) Protein expression of granzyme B (WT: n = 8; MD4: n = 6; μMT: n = 7; IgMi: n = 8) and TNF-α (WT: n = 7; MD4: n = 7; μMT: n = 8; IgMi: n = 7) from BALF. Data are represented as mean ± SEM, and P values were determined by repeated 1-way ANOVA measures (*P < 0.05, **P < 0.01, ***P <0.001, ****P < 0.0001).

Figure 5. Loss of influenza virus–specific antibody increases lung tissue injury during memory superinfection.

Histology scoring and QuPath analysis were performed on lungs from WT, μMT, MD4, and IgMi mice. (A) Right: Representative H&E lung images, scanned at ×40. Left: A heatmap overlay was used to signify lung areas with inflammation/consolidation. (B) Representative histology sections with H&E of WT, μMT, MD4, and IgMi lungs. Scale bars: 200 μm. (C) Histology scores of perivascular lung tissue sections. (D and E) Quantification of lung nucleated cell density (cells/mm²) and frequency of lung inflammation and consolidation detections (WT: n = 17; MD4: n = 11; μMT: n = 17; IgMi: n = 8). (F) BALF protein at day of harvesting (WT: n = 30; MD4: n = 10; μMT: n = 32; IgMi: n = 7). Data are represented as mean ± SEM, and P values were determined by repeated 1-way ANOVA measures (*P < 0.05, **P < 0.01, ****P < 0.0001).

Figure 6. Temporal B cell depletion during primary influenza virus infection leads to increased lung memory CD8⁺ T cell formation.

(A) B cell depletion scheme. B cells repopulated the lung before challenge with PR8 followed by MRSA. IT, intratracheal. (B) Depletion efficiency in the lung was confirmed via flow cytometry (day 0, isotype control [ISO]: n = 4; day 0, B cell–depleted: n = 4; day 30, ISO: n = 4; day 30, B cell–depleted: n = 3). (C) X-31 influenza virus–specific mouse serum titers measured by hemagglutinin inhibition assay (HAI) (ISO: n = 7; B cell–depleted: n = 5). (D) Percentage of weight loss for each group (ISO: n = 14; B cell–depleted: n = 12). (E and F) Histology scores of parenchymal lung sections and quantification of lung nucleated cell density (cells/mm²) (ISO: n = 8; B cell–depleted: n = 7). (G) MRSA colonies from lung homogenates (ISO: n = 6; B cell–depleted: n = 5). (H–J) Percentage of lung CD8⁺ cells, CD8⁺CD69⁺CD103^hi cells, and PD-1⁺CD8⁺ cells (ISO: n = 14; B cell–depleted: n = 11). (K) BALF granzyme B protein concentration (ISO: n = 6; B cell–depleted: n = 5). Data are represented as mean ± SEM, and P values were determined by repeated 2-tailed Mann-Whitney U test (*P < 0.05, **P < 0.01, ***P <0.001, ****P < 0.0001).

Figure 7. Passive immunization of μMT mice with heterotypic memory influenza serum rescues weight loss and tissue injury.

WT or μMT mice were intravenously injected with 150 μL of either PBS vehicle, pooled naive WT serum, or pooled WT memory serum after PR8 challenge (day 54). (A) Weight loss for each group. (B) Flow cytometry was performed to calculate absolute number of lung CD8⁺CD45⁺CD90.2⁺ cells. (C and D) Percentage of effector memory CD44^hiCD62^loCD8⁺ cells and PD-1⁺CD8⁺ cells. (E) Absolute number of CD64⁺CD24^–Ly6G^–CD45⁺CD19^–TCRβ^– cells. (F) Histology scoring of perivascular lung sections. (G) BALF granzyme B protein concentration. (For A–G: WT-vehicle: n = 12; WT–memory Ab: n = 8; WT–naive Ab: n = 8; μMT-vehicle: n = 10; μMT–memory Ab: n = 8; μMT–naive Ab: n = 8). Data are represented as mean ± SEM, and P values were determined by repeated 1-way ANOVA measures (*P < 0.05, **P < 0.01, ***P <0.001, ****P < 0.0001).

Figure 8. CD8⁺ T cells induced by B cell deficiency promote bacterial control during memory superinfection.

(A) CD8⁺ T cell depletion scheme. IT, intratracheal. (B) CD8β T cell depletion was confirmed on day of tissue harvesting via flow cytometry (μMT–CD8β-depleted: n = 8; WT–CD8β-depleted: n = 8; μMT-ISO: n = 8; WT-ISO: n = 8). (C) MRSA burden in lung homogenates (μMT–CD8β-depleted: n = 8; WT–CD8β-depleted: n = 8; μMT-ISO: n = 7; WT-ISO: n = 7). (D) Viral protein (PR8) was assessed via quantitative PCR (μMT–CD8β-depleted: n = 8; WT–CD8β-depleted: n = 8; μMT-ISO: n = 8; WT-ISO: n = 8). (E) BALF granzyme B protein concentration (μMT–CD8β-depleted: n = 8; WT–CD8β-depleted: n = 8; μMT-ISO: n = 8; WT-ISO: n = 8). (F) WT and μMT mice were challenged with a lethal dose of MRSA (2 × 10⁸) during memory superinfection and weighed daily (left). PR8-induced weight loss prior to MRSA challenge (day 54 to day 60) (right), and percentage of weight loss 48 hours following MRSA challenge (day 62) (middle) (WT: n = 22; μMT: n = 21). (G) Left: Survival percentage was calculated daily after lethal MRSA challenge. Right: Median day of mortality was calculated for WT and μMT groups (WT: n = 22; μMT: n = 21). (H) MRSA burden 14 hours after lethal MRSA challenge in lung homogenate (WT: n = 14; μMT: n = 13). Data are represented as mean ± SEM. For B–E, P values were determined by repeated 1-way ANOVA measures; for F–H, P values were determined by repeated 2-tailed Mann-Whitney U test (*P < 0.05, **P < 0.01, ***P <0.001, ****P < 0.0001).