Mitochondria maintain controlled activation state of epithelial-resident T lymphocytes

Abstract

Epithelial-resident T lymphocytes, such as intraepithelial lymphocytes (IELs) located at the intestinal barrier, can offer swift protection against invading pathogens. Lymphocyte activation is strictly regulated because of its potential harmful nature and metabolic cost, and most lymphocytes are maintained in a quiescent state. However, IELs are kept in a heightened state of activation resembling effector T cells but without cytokine production or clonal proliferation. We show that this controlled activation state correlates with alterations in the IEL mitochondrial membrane, especially the cardiolipin composition. Upon inflammation, the cardiolipin composition is altered to support IEL proliferation and effector function. Furthermore, we show that cardiolipin makeup can particularly restrict swift IEL proliferation and effector functions, reducing microbial containment capability. These findings uncover an alternative mechanism to control cellular activity, special to epithelial-resident T cells, and a novel role for mitochondria, maintaining cells in a metabolically poised state while enabling rapid progression to full functionality.

Fig. 1.. IELs have a heightened metabolic state.

(A) Memory CD8⁺ T cells from spleen or intestinal IEL fraction were isolated by flow-sorting (purity, >98%) and differential expression (DESeq) analysis of gene expression was performed. Heatmap of normalized gene expression of differentially expressed mRNAs associated with lipid metabolism is shown (n = 3). (B and C) Flow-sorted CD8⁺ T cells sourced from the spleen (CD44^lo, naive; CD44^hi, memory) or intestinal IEL fraction were stained with Nile Red. Cells were analyzed by (B) fluorescence microscopy counterstained with DAPI in blue. Representative overview (left column) and closeup (right column) pictures of more than four biological repeats, or (C) flow cytometry (n = 4) for mean fluorescence intensity (MFI) of Nile Red. a.u., arbitrary units; ns, not significant. Scale bars, 5 μm. (D) Naive CD8⁺ T cells were purified by magnetic cell sorting and stimulated with plate-bound anti-CD3/CD28 for 2 days, cells were maintained in IL-2 (diamonds) and analyzed daily by flow cytometry, and Nile Red fluorescence intensity levels were plotted (n = 3; three samples were analyzed in this experiment shown, representative of three biological repeats). Ex vivo isolated IELs (triangle) assessed on the same day are shown. For statistical analysis, one-way ANOVA with multiple comparison test was used. Error bars indicate SDs. **P < 0.01, ***P < 0.001.

Fig. 2.. IELs are in heightened activation state.

(A and B) CD8⁺ T cells obtained from the spleen (CD44^lo, naive; CD44^hi, memory) or small intestinal IEL fraction were stained intracellular for (A) LAMP-1 (CD107a) and (B) Lysotracker (n = 4). MFIs are shown. (C) Differential expression (DESeq, Negative Binomial Distribution) analysis comparing flow cytometry-sorted spleen sourced CD8⁺ memory cells (CD44^hi) and small intestinal CD8⁺ IELs. Heatmap of normalized gene expression of genes associated with cytotoxic potential in T cells is shown (n = 3). (D) Representative EM pictures of flow-sorted CD8⁺ T cells; naive and memory T cells from the spleen and IELs. Indicated with arrows are secretory granules. For statistical analysis, one-way ANOVA with multiple comparison test was used. Error bars indicate SDs. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.. IELs are metabolically arrested.

(A) Flow-sorted CD8⁺ T cells were processed for imaging with TEM, and the number of mitochondria per cell was quantified (n = 10). (B to E) Naive CD8⁺CD44^lo or memory CD8⁺CD44^hi T cells obtained from spleens and IELs obtained from the small intestine were flow-sorted (>98% purity) and assessed on a Seahorse analyzer. (B) Extracellular flux analysis comparing CD8⁺ T cell subsets sourced from the spleen (CD44^lo naive; CD44^hi, memory; naive T cells after 24 hours of anti-CD3/CD28 stimulation, effector) and IELs from the small intestine. OCR is plotted against time under basal conditions and in response to indicated chemicals. Oligo, oligomycin; Rot, rotenone; Ant, antimycin A. (C) SRC (maximum OCR/baseline OCR) for indicated cell subsets. (D) ECAR for indicated CD8⁺ T cell populations. (E) As described in (B), but upon flow-sorting of the two major IEL populations, via negative sorting based on TCR identity, TCRαβ or TCRγδ T cells were assessed separately compared with memory T cells. Data are representative of at least two experiments performed in triplicate. For statistical analysis, one-way ANOVA with multiple comparison test was used. Error bars indicate SDs. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4.. IELs have altered mitochondria.

(A and B) Flow-sorted CD8⁺ T cells sourced from the spleen (CD44^lo, naive; CD44^hi, memory) or the small intestinal IEL fraction were stained with MitoTracker Green. Cells were analyzed by (A) fluorescence microscopy counterstained with DAPI in blue (representative picture of four independent biological repeats) or (B) flow cytometry (n = 3 to 4) for MFI of MitoTracker Green. Scale bars, 5 μm. (C) Naive CD8⁺ T cells were purified by magnetic cell sorting and stimulated with plate-bound anti-CD3/CD28 for 2 days, and cells were maintained in IL-2 (diamonds). Cells were analyzed daily by flow cytometry, and MitoTracker fluorescence intensity levels were plotted. Freshly obtained intestinal IELs were analyzed simultaneously (n = 3; three biological repeats), and average values were plotted as a triangle. (D to F) Mitochondrial membrane potential (MTO M7510) (D) (pooled data from two experiments with n = 3), mitochondrial (MitoSOX) (E), or total cellular (H₂DCFDA) ROS production (F) (pooled data from two experiments with n = 2 to 3). For statistical analysis, one-way ANOVA with multiple comparison test was used. Error bars indicate SDs. **P < 0.001,***P<0.001.

Fig. 5.. IEL mitochondrial staining correlates with activation.

(A) IELs were purified by flow cytometry and transferred intravenously into lymphopenic hosts. After 6 weeks, MitoTracker Deep Red-based detection of mitochondria was performed in indicated cell populations [trIELs (transferred IEL)] (pooled data from two experiments with n = 3). (B) C57BL/6 mice were infected intragastrically with 1000 E. vermiformis oocysts, and MitoTracker Green-based detection of mitochondria was performed on IELs harvested at indicated time points after infection from the ilea (n = 5). dpi, days post infection. (C) IELs were purified from control mice and IL-22-deficient mice treated or not with broad-spectrum antibiotics (ABX) and mitochondria assessed with MitoTracker Green (pooled data from two experiments with n = 4). (D to I) C57BL/6 mice were intraperitoneally injected with anti-CD3, and (D) MitoTracker Green-based detection of mitochondrial mass was assessed at indicated time points (pooled data from three experiments with n = 2 to 3). (E) Proportion of IELs positive for Ki67 and (F) number of T cells by flow cytometry analysis at indicated time points after anti-CD3 administration (representative of three biological repeats, n = 4 to 5). MitoTracker Green-based detection of mitochondria was performed in indicated cell populations, naive and memory CD8⁺ cells from the spleen, and TCRαβ (G) and TCRγδ5 (H) IELs isolated from the small intestine at indicated time points after anti-CD3 injection (representative of more than three biological repeats, n = 5 to 10). (I) Mitochondrial membrane potential (MTO M7510) on indicated CD8⁺ T cell populations at indicated time points after anti-CD3 injection (pooled data from two experiments). One-way ANOVA with multiple comparison test was used. Error bars represent SDs. **P < 0.01, ***P < 0.001.

Fig. 6.. IEL mitochondria have altered CL makeup.

(A) Flow-sorted CD8⁺ T cells were processed for imaging with TEM, and mitochondrial surface area was determined for each mitochondrion within a given cell. Data are represented as an average for mitochondrial surface area on a per-cell basis (n = 10 naive cells, n = 10 memory cells, and n = 12 IELs). (B and C) CD8⁺ T cells from spleen or intestinal IELs were stained with NAO in (B) steady state (n = 3 from three biological repeats) or (C) indicated CD8⁺ T cell populations at indicated time points after anti-CD3 injection (pooled data from three experiments with n = 2 to 3). (D and E) Flow-sorted naive and memory CD8⁺ T cells, as well as IELs, were analyzed by mass spectrometry for (D) their total CL content (n = 9) or (E) the relative contribution of selected CL species (n = 8). One-way ANOVA with multiple comparison test was used. Error bars represent SDs. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 7.. Altered CL makeup of IEL mitochondria correlates with IEL function.

(A to D) C57BL/6 mice were infected intragastrically with 1000 E. vermiformis oocysts, and MitoTracker Green-based detection of mitochondria was performed on ilea-sourced IELs harvested 10 days after infection (≥4 biological repeats, n ≥ 2 per experiment) or from uninfected controls. MFI of MitoTracker Green staining was plotted (x axis) against (A) Ki67, (B) mitochondrial ROS production, and (C) proportion of IFNγ or (D) TNF production. (E) Mass spectrometry analysis of the relative contribution of selected CL species detected in indicated CD8⁺ T lymphocytes (n = 4 to 5). Act, activated. (F) ROS production by MitoSOX staining was performed on mice injected with anti-CD3 at indicated time points (pooled data from three experiments with n ≥ 3). For statistics, linear regression was used for (A) to (D) and one-way ANOVA with multiple comparison test was used for (E) and (F). Error bars represent SDs. ***P < 0.001.

Fig. 8.. Altered CL directly affects IEL function.

C57BL/6 Rag2-deficient mice were reconstituted with wild-type control (closed symbols) or Tafazzin-deficient (open symbols) bone marrow. (A to J) Six to 8 weeks afterward, mice were injected intraperitoneally with anti-CD3 and CD8⁺ T cells from the intestinal IEL compartment (A and C) or the spleen (B) were assessed at indicated time points for (A) MitoTracker Green or (B and C) Ki67 (three biological repeats with n = 2 to 3). Six to 8 weeks after bone marrow chimeric generation, mice were challenged with 1000 Ev oocysts, and (D) parasite load was assessed for the first 10 days daily or (E) accumulative load of shedded oocysts (accumulated from two biological repeats, n = 5 to 6). At day 10 postinfection, cells from the ileum IEL compartment were stained for Ki67 in (F) CD4⁺ T cells, (G) CD8αβ⁺ T cells, and (H) CD8αα⁺ T cells. Furthermore, the IFNγ production in (I) CD8⁺ or (J) CD4⁺ T cells was assessed via intracellular staining (representative of two biological repeats with n = 5). Error bars indicate SDs. For statistics, multiple t test was used. *P < 0.05, **P < 0.01, ***P < 0.001.