Tissue-resident CD4+ T helper cells assist the development of protective respiratory B and CD8+ T cell memory responses

Abstract

Much remains unknown about the roles of CD4⁺ T helper cells in shaping localized memory B cell and CD8⁺ T cell immunity in the mucosal tissues. Here, we report that lung T helper cells provide local assistance for the optimal development of tissue-resident memory B and CD8⁺ T cells after the resolution of primary influenza virus infection. We have identified a population of T cells in the lung that exhibit characteristics of both follicular T helper and T_RM cells, and we have termed these cells as resident helper T (T_RH) cells. Optimal T_RH cell formation was dependent on transcription factors involved in T follicular helper and resident memory T cell development including BCL6 and Bhlhe40. We show that T_RH cells deliver local help to CD8⁺ T cells through IL-21-dependent mechanisms. Our data have uncovered the presence of a tissue-resident helper T cell population in the lung that plays a critical role in promoting the development of protective B cell and CD8⁺ T cell responses.

Fig. 1.. Lung CD4⁺ T cells deliver localized help to B and CD8⁺ T cells.

(A to D) WT mice were infected with PR8 strain of influenza virus and treated with control (Ctl) IgG or α-CD4 starting at 14 d.p.i. Mice were injected with α-CD45 intravenously 5 min before sacrifice at 42 d.p.i. (A) Experimental scheme. (B) Representative confocal images of iBALT in the lung. Lung sections were stained with α-CD4 (red), α-B220 (green), and DAPI (blue). (C) Frequencies and cell number of influenza HA-B cells in the lung tissue or blood vessels. (D) Lung tissue CD8⁺, CD8⁺CD69⁺, or CD8⁺CD69⁺CD103⁺ NP_366–374 or PA_224–233 T_RM cells were enumerated. (E to I) WT mice were infected with PR8 and treated with Ctl IgG or α-CD4 (starting at 14 d.p.i.) in the presence of daily injection of FTY720 (starting at 13 d.p.i.). (E) Schematic of experimental design. (F) B_GC, (G) total HA-B (CD45_i.v.⁻HA-B), or (H) HA-B_RM cells were enumerated by flow cytometry. (I) Lung tissue CD8⁺, CD8⁺CD69⁺, or CD8⁺CD69⁺CD103⁺ NP_366–374 T_RM cells were enumerated. (J to L) WT mice were infected with PR8 and received Ctl IgG and high or low dose of α-CD4. (J) Experimental scheme. (K) Cell number of B_GC, HA-B_RM, or NP-B_RM cells in the lung tissue. (L) Lung parenchymal CD8⁺, CD8⁺CD69⁺, or CD8⁺CD69⁺CD103⁺ NP_366–374 T_RM cells were enumerated. In (B) to (D) were the representative data from at least two independent experiments (four to five mice per group). In (F), (G), and (I) to (L), data were pooled from two (F to G and I) or three (K and L) independent experiments (two to five mice per group). P values were calculated by unpaired two-tailed Student’s t test in (C), (D), and (F) to (I). P values in (J) and (K) were analyzed by one-way ANOVA. Data are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. n.s., not significant.

Fig. 2.. Identification of a population of T_FH-like cells in the lung tissue.

WT (A to E) or IL-21-VFP reporter (F) mice were infected with PR8. (A) t-Distributed stochastic neighbor embedding (tSNE) plot of scRNA-seq analysis of sorted lung CD45_i.v.⁻CD4⁺CD44^Hi cells (pooled from five mice) at 28 d.p.i. (B) Heat map of indicated genes in each cluster from scRNA-seq data. (C) Kinetics of the percentages of PD-1^HiFR4^Hi population in lung tissue, total CD4⁺, or influenza NP-specific (NP_311–325) CD4⁺ T cells. (D) Expression of T_FH cell–associated markers in lung total or influenza-specific PD-1^HiFR4^Hi, PD-1^LowFR4^Low, or splenic T_FH (CD4⁺CD44^HiPD-1⁺CXCR5⁺) cells at 28 d.p.i. (E) Frequency positive for IFN-γ, IL-17, or IL-4 by lung PD-1^HiFR4^Hi, PD-1^LowFR4^Low, or spleen T_FH cells were identified by intracellular staining at 28 d.p.i. (F) IL-21-VFP expression in lung CD4⁺ PD-1^HiFR4^Hi, PD-1^LowFR4^Low, or spleen T_FH at 28 d.p.i. In (C) to (F), representative plots, histograms, and graphs were collected from at least two independent experiments (two to four mice per group). P values in (E) and (F) were analyzed by one-way ANOVA. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 3.. Transcriptional profiling reveals PD-1^HiFR4^Hi cells exhibit both T_FH and T_RM gene signatures.

(A to F) WT mice were infected with PR8. Lung PD-1^HiFR4^Hi or PD-1^LowFR4^Low CD4⁺ T cells and splenic T_FH or non-T_FH cells were sorted after exclusion of GITR^Hi T_reg cells, and RNA-seq analysis was performed at 28 d.p.i. (A) Heatmap of DEGs among lung PD-1^HiFR4^Hi, PD-1^LowFR4^Low CD4⁺ T cells, and splenic T_FH or non-T_FH cells. (B) Principal component analysis (PCA) of RNA-seq data of lung PD-1^HiFR4^Hi, PD-1^LowFR4^Low CD4⁺ T cells, and splenic T_FH or non-T_FH cells. (C) Volcano plot of RNA-seq analysis of lung PD-1^HiFR4^Hi or PD-1 ^LowFR4^Low CD4⁺ T cells. (D) GSEA of the core T_FH signature genes in lung CD4⁺ PD-1^HiFR4^Hi and CD4⁺ PD-1^LowFR4^Low cells. (E) Volcano plot of RNA-seq analysis on lung PD-1^HiFR4^Hi CD4⁺ T and splenic T_FH cells. (F) GSEA of the core tissue residency signature genes of T_RM cells in lung PD-1^HiFR4^Hi and splenic T_FH cells. (G and H) WT mice were infected with PR8. Lung PD-1^HiFR4^Hi or PD-1^LowFR4^Low CD4⁺ T cells and splenic T_FH cells were sorted at 28 d.p.i. Nanostring analysis on 560 immune-associated genes was performed. The expression of T_FH-associated genes (G) or tissue residency–associated genes (H) in the three cell populations was depicted. For RNA-seq, data were from duplicates of pooled samples (n = 15). For nanostring analysis, data were from pooled samples (n = 10). ES, enrichment score; NES, normalized enrichment score; FDR, false discovery rate.

Fig. 4.. Lung PD1^HiFR4^Hi CD4⁺ T cells are tissue resident.

(A) WT mice were infected with PR8. The expression of CD69, CXCR6, and Bhlhe40 in lung CD4⁺ PD-1^HiFR4^Hi, CD4⁺ PD-1^LowFR4^Low NP_311–325 T cells, or splenic T_FH cells at 28 d.p.i. (B to E) CD45.1⁺ (host) or CD45.1⁺ CD45.2⁺ (partner) WT mice were infected with PR8. Parabiosis surgery was performed at 21 d.p.i. Mice were euthanized 14 days later for analysis. (B) Schematic of parabiosis experiments. (C) Composition of Host-derived or Partner-derived CD4⁺ T cells in the spleen of the parabionts. (D) Frequencies of Host-derived or Partner-derived cells in the lung PD-1^HiFR4^Hi or PD-1^LowFR4^Low total CD4⁺ T cell compartment. (E) Frequencies of Host-derived or Partner-derived cells in influenza-specific lung CD4⁺ PD-1^HiFR4^Hi or CD4⁺ PD-1^LowFR4^Low NP_311–325 T cell compartment. In (A), the representative histograms were from at least two independent experiments (three to four mice per group). In (C) to (E), parabiosis data were pooled from two different experiments. P values were calculated by one-way ANOVA in (D) and (E). Data are means ± SEM. **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 5.. Both BCL6 and Bhlhe40 are required for optimal lung T_RH cell responses.

(A to D) Bcl6^fl/fl or Bcl6^ΔT mice were infected with PR8. (A) Representative dot plot, (B) frequencies, and (C) cell numbers of T_RH or non-T_RH in lung CD45_i.v.⁻ total CD4⁺ or CD45_i.v.⁻ influenza-specific CD4⁺ NP_311–325 T cells at 28 d.p.i. (D) Frequencies (top) or cell numbers (bottom) of splenic total T_FH or NP_311–325 T_FH at 28 d.p.i. (E) GSEA of the Bhlhe40-associated genes in lung T_RH (PD-1^HiFR4^Hi) and spleen T_FH cells. (F to H) Bhlhe40^fl/fl or Bhlhe40^ΔT mice were infected with PR8. (F) Representative dot plot, (G) frequencies (top), or cell numbers (bottom) of lung NP_311–325 T_RH or non-T_RH cells at 28 d.p.i. (H) Active caspase 3/7–fluorochrome-labeled inhibitors of caspases (FLICA)⁺ cells in lung NP_311–325 T_RH or non-T_RH cells at 28 d.p.i. In (A) to (D) and (F) to (G), data were pooled from two independent experiments (three to four mice per group). In (H), representative data were from at least two independent experiments (four mice per group). P values were calculated by unpaired two-tailed Student’s t test. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 6.. T_RH cells are required for the development of lung protective CD8⁺ T_RM and B cell immunity.

(A to J) Bcl6^fl/fl or Bcl6^ΔCD4ERT2 mice were infected with PR8. (A to E) Tamoxifen was administrated daily (12 to 16 d.p.i.) in the presence of daily FTY720 administration (11 to 34 d.p.i.). (A) Schematic of experimental design. Cell numbers of (B) B_GC, (C) HA-B_RM, (D) NP-B_RM, (E) CD8⁺CD69⁺ NP_366–374 T_RM, or CD8⁺CD69⁺ PA_224–233 T_RM at 35 d.p.i. (F to J) Tamoxifen was administrated daily from 21 to 25 d.p.i. in the presence of daily FTY720 administration (20 to 41 d.p.i.). (F) Schematic of experimental design. Cell numbers of (G) B_GC, (H) HA-B_RM, (I) NP-B_RM, (J) CD8⁺CD69⁺ NP_366–374 T_RM, or CD8⁺CD69⁺ PA_224–233 T_RM at 42 d.p.i. (K) Bcl6^fl/fl or Bcl6^ΔCD4ERT2 mice were infected with X31 strain (H3N2) of influenza. Tamoxifen was administrated daily (12 to 16 d.p.i.) in the presence of daily FTY720 administration (11 to 34 d.p.i.). Representative dot plot (left) and cell numbers (right) of X31 strain-specific B_RM or cross-reactive HA-B_RM (to H3N2 A/Uruguay/716/07 strain) at 35 d.p.i. (L and M) Bcl6^fl/fl (n = 11) or Bcl6^ΔCD4ERT2 (n = 15) mice were infected with X31 and administered tamoxifen from 12 to 16 d.p.i. Mice were rechallenged with PR8 at 42 d.p.i. in the presence of FTY720 (starting from 41 days). (L) Schematic of experimental design. (M) Host mortality following PR8 challenge was monitored. (N and O) Bcl6^fl/fl or Bcl6^ΔCD4ERT2 mice were infected with X31 and administered tamoxifen. Mice were challenged with PR8 at 40 d.p.i. in the presence of FTY720. Lung (N) infectious virus titers and (O) viral gene expression were measured at day 2 post rechallenge. In (A) to (H) and (J) to (K), all data were pooled from two (C, D, G, H, J, and K) or three (B and E) independent experiments (two to five mice per group). In (A) to (O), P values were calculated by unpaired two-tailed Student’s t test. P value of survival study in (M) was calculated by log-rank test. Data are means ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001.

Fig. 7.. T_RH help to CD8⁺ T cells is IL-21 dependent.

(A) Representative dot plot of IL-21^Hi or IL-21^Low CD4⁺ T cells from the IL-21-VFP reporter mice infected with PR8 (28 d.p.i.). (B to E) WT mice were infected PR8 with or without IL-21R blockade through intraperitoneal (i.p.) route starting at 14 d.p.i. in the presence of FTY720 administration (13 to 34 d.p.i.). (B) Experimental scheme. Cell numbers of lung parenchymal (C) B_GC, (D) HA-B_RM, and (E) CD8⁺CD69⁺ NP_366–374, or CD8⁺CD69⁺ PA_224–233 T_RM cells. (F to J) WT mice were infected with PR8 with or without IL-21R blockade through intranasal (i.n.) route at 14 d.p.i. (F) Experimental scheme. (G) Frequencies or (H) cell numbers of lung tissue CD8⁺CD69⁺ NP_366–374 or CD8⁺CD69⁺ PA_224–233 T_RM. (I) Percentage of apoptotic cells were identified by active caspase 3/7-FLICA staining within lung CD8⁺ NP_366–374 T_RM or splenic CD8⁺ NP_366–374 T_MEM at 28 d.p.i. (J) Representative histogram of BATF expression in lung CD8⁺ NP_366–374 or CD8⁺ PA_224–233 T_RM of mice received with Ctl IgG or α-IL21R at 35 d.p.i. FMO, fluorescence minus one control. (K to M) WT mice were infected with PR8 with or without IL-21R blockade in the presence of FTY720. Mice were challenged with X31 at 40 d.p.i. (K) Experimental scheme. (L) Lung infectious virus titer or (M) viral gene expression were measured at day 2 after rechallenge. In (A) and (I) to (M), representative data were from at least two independent experiments (four to five mice per group). In (C), (E), and (G) to (H), data were pooled from two independent experiments (three to five mice per group). P values of all experiments were calculated by unpaired two-tailed Student’s t test. Data are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.