Research ArticleTRANSPLANTATION

Generation and persistence of human tissue-resident memory T cells in lung transplantation

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Science Immunology  08 Mar 2019:
Vol. 4, Issue 33, eaav5581
DOI: 10.1126/sciimmunol.aav5581
  • Fig. 1 Donor-derived memory T cells persist specifically within the lung allograft.

    Donor- and recipient-derived T cells were evaluated in blood and BAL samples of lung transplant recipients by flow cytometry based on HLA class I disparities (see Materials and Methods). (A) Schematic of experimental design to follow how donor- and recipient-derived T cells would interact in lung transplant recipients. (B) Representative flow cytometry plots of donor versus recipient CD4+ (left) and CD8+ (right) T cells derived from peripheral blood. (C) Representative flow cytometry plots show CD4+ (middle) and CD8+ (right) T cell frequency and donor/recipient origin from a representative BAL sample. (D) Left: Graphs show percent CD4+ (top) and CD8+ (bottom) T cells of donor origin (relative to total CD4+ or CD8+ T cells) in peripheral blood over time after transplantation in individual patients (n = 14 patients with more than three samples over time). Right: Absolute cell counts of donor CD4+ (top) and CD8+ T cells (bottom) in peripheral blood in the same patients, with dotted lines representing average recipient T cell count over time. (E) Graphs show percent CD4+ (top) and CD8+ (bottom) T cells of donor origin (relative to total CD4+ or CD8+ T cells) in BAL samples after transplantation, showing individual curves for each of 20 patients. Symbols for each individual patient are designated in the legend at the right. (F) CD4:CD8 T cell content in the BAL of transplant recipients (total T cell content from donor and recipient) at indicated times after transplantation. Results are shown for 19 patients; ***P < 0.01; all other comparisons are nonsignificant.

  • Fig. 2 BAL of human lungs samples T cells from both the lung parenchyma and airways.

    (A) Schematic diagram (left) highlighting the lateral basilar segment of the lower lobe, which is where the BAL was performed and where control lung and airway segments were procured, and photograph (right) of one study lung included in the analysis. (B) Representative flow cytometry plots showing CD4 and CD8 ratio across locations. ***P = 0.0008. (C) T cell subset composition in the BAL, airway, and lung parenchyma showing effector memory (TEM; CCR7CD45RA), terminal effector (TEMRA; CCR7CD45RA+), central memory (TCM; CCR7+CD45RA), and naïve T cells (CCR7+CD45RA+) in representative flow cytometry plots (left) and compiled frequencies (means ± SEM) from 15 control lungs. **P = 0.006. (D) Cell surface expression of tissue residency markers CD69 and CD103 by CD4+ and CD8+ T cells across locations shown as representative flow cytometry plots (left) and compiled frequencies (right) from 15 donors. **P = 0.0013, ***P = 0.0002, and ****P < 0.0001. ns, not significant.

  • Fig. 3 Donor and recipient BAL T cells are phenotypically and functionally memory T cells.

    (A) Subset composition of donor and recipient CD4+ and CD8+ T cells in representative patient BAL based on CD45RA and CCR7 expression as in Fig. 2. (B) Compiled data (means ± SEM) of donor (left) and recipient (right) T cell subset composition (TEM, blue; TEMRA, red; näive, green; TCM, black) over time after transplantation for CD4+ (top) and CD8+ T cell (bottom) lineages. Results compiled from 20 patients with one sample per patient per time point. (C) HLA-DR expression by donor and recipient T cells in patient BAL at indicated times after transplantation shown as representative flow cytometry plots (left) and compiled frequencies from 23 samples derived from 11 patients (right). (D to F) T cells from patient BAL samples were stimulated with PMA/ionomycin, and cytokine production was determined by intracellular staining after 5 hours. (D) IFN-γ production by donor (black) and recipient (red) CD4+ TEM (left) and CD8+ TEM (right) in representative flow cytometry plots and graphs showing paired frequencies (based on percent cytokine+ of donor or recipient T cells) in patient BAL samples (n = 8) 1 to 9 months after transplantation (right). *P = 0.04 for CD4+ TEM and *P = 0.01 for CD8+ TEM. (E) IL-17 production by donor (black) and recipient (red) CD4+ TEM in representative flow cytometry plot (left) and graph (right) showing paired frequencies in patient BAL samples (n = 7) 1 to 9 months after transplantation. *P = 0.05. (F) IL-2 production by donor (black) and recipient (red) CD4+ TEM in representative flow cytometry plot (left) and graph showing paired frequencies in patient BAL samples (right; n = 8). (G) Granzyme B (GzB) expression by CD4+ TEM (left) and CD8+ TEM (right) from BAL samples obtained >2 months after transplantation in representative flow cytometry plots (left) and cumulative paired data (right) from six transplant recipients of donor (black) and recipient (red). *P = 0.04.

  • Fig. 4 Differential expression of TRM markers by donor- and recipient-derived T cells with time.

    (A) Expression of CD69 and CD103 by CD4+ (top two rows) and CD8+ T cells (third and fourth rows) of donor or recipient origin as indicated over time after transplantation for one representative patient, P5. (B) Graphs show mean frequency (±SEM) of CD69 expression (left) or CD69/CD103 coexpression (right) by CD4+ TEM (top) or CD8+ TEM (bottom) cells in patient BAL at indicated times after transplantation, compiled from 20 patients. T cell origin designated as donor (blue) and recipient (red) based on HLA disparities (see Fig. 1); gray shaded rectangles denote 1 SD around the average frequency of CD69+ (left) and CD69+CD103+ (right) expression by control BAL T cells compiled from Fig. 2D. Expression of (C) CD49a, (D) PD-1, and (E) CD101 by BAL, airways, and parenchyma obtained from control lungs shown as representative flow cytometry plots and mean frequencies (±SEM), compiled from 14 lungs. *P = 0.02 (CD101); *P < 0.05 and **P = 0.004 (CD49a); ns (PD-1). (F) CD49a expression by donor (black) and recipient (red) CD4+ TEM (top) and CD8+ TEM (bottom) cells in patient BAL samples shown as representative flow cytometry plots (left) and in graphs showing paired frequencies from individual patient BAL samples (n = 6) at >1 month after transplantation. ***P = 0.0003 and *P = 0.007. (G) PD-1 expression by donor (black) and recipient (red) CD4+ TEM (top) and CD8+ TEM (bottom) cells in patient BAL shown as representative flow cytometry plots (left) and graphs showing paired frequencies in patient BAL samples (n = 14) >6 months after transplant. ***P = 0.0001. (H) CD101 expression by donor (black) and recipient (red) CD4+ TEM (top) and CD8+ TEM (bottom) cells in representative flow cytometry plots (left) and paired frequencies in patient BAL samples (n = 15) at >1 month after transplantation. *P = 0.02.

  • Fig. 5 Single-cell transcriptome profiling of BAL T cells reveals three distinct subsets with differential expression of TRM-associated genes.

    (A) Representative flow cytometry plots from the BAL of P29 11 months after transplantation showing gating strategy to identify cell populations sorted into 96-well plates for scRNA-seq; “***” indicates the population sorted: live, CD3+, lymphocytes, and pan-HLA+. (B) Representative flow cytometry plot from influx sorter identifying cell surface markers indexed to individual wells. (C) PC clustering identifying three distinct clusters demarcated by color and tSNE plots visualizing cluster differentiation (see Materials and Methods); indexed cell surface markers identifying cell origin (donor versus recipient) and protein surface expression for CD4, CD8, CD69, and CD103 are indicated within each cluster in separate tSNE plots. (D) Heat map illustrating single-cell analysis for top differentially expressed genes along with select genes of interest, arranged by cluster. Heat map of z-scored expression values, where expression values are defined as log(1+nUMIijmedian(nUMIj)nUMIj), where nUMIij is the number of UMI counts for gene i in cell j, and nUMIj is the total number of UMI counts in cell j.

  • Fig. 6 TRM-like subsets exhibit quantitative gene expression differences in donor and recipient T cells and are consistent between patients.

    (A) Heat map of the z-scored mean expression values (as defined in Fig. 5D) of select genes based on differential expression analysis in each cluster, with cluster 1 designated as mature TRM, cluster 2 designated as TEM, and cluster 3 designated as TRM-like. (B) Volcano plots showing differential gene expression in donor compared with recipient T cells in each cluster. (C) PC clustering from scRNA-seq analysis of CD3+ T cells obtained from the BAL of P19 13 months after transplantation identifies two distinct clusters, demarcated by color; tSNE plots visualizing cluster differentiation. (D) Pearson correlation analysis comparing gene expression within clusters 1, 2, and 3 from patient P29 to gene expression within clusters 1 and 2 from patient P19.

  • Fig. 7 Donor- and recipient-derived T cells cluster near airways.

    Immunofluorescence imaging of transbronchial biopsies (TBBx) obtained from three transplant recipients (P12, P20, and P23); for P12 and P20, the recipient is HLA-A2+ and donor is HLA-A2; for P23, the recipient is HLA-A2 and donor is HLA-A2+. Short yellow arrows point to recipient-derived T cells, and long green arrows point to donor-derived T cells. (A, D, and G) Hematoxylin and eosin (H&E)–stained samples showing small airway with cluster of lymphocytes. (B, E, and H) Expression of E-cadherin (purple), DAPI (blue), CD4 (green), and HLA-A2 (red). (C, F, and I) Expression of E-cadherin (purple), DAPI (blue), CD8 (green), and HLA-A2 (red).

  • Fig. 8 Donor TRM persistence is associated with reduced clinical complications.

    Patient records were examined for clinical complications including PGD and episodes of ACR at all time points of BAL acquisition (see Materials and Methods). (A) Graphs show percentage (mean ± SEM) of donor CD4+ (left) or CD8+ (right) T cells in the BAL over indicated times after transplantation in patients (n = 20) stratified on the basis of those who experienced PGD (red) or did not (blue). **P = 0.003 (left) and **P = 0.002 (right). Cumulative data across all time points showed increased proportion of donor CD8+ (P = 0.008) and trends in increased donor CD4+ (P = 0.06) T cells in those participants without PGD. (B) Donor CD4+ (left) and CD8+ (right) T cell frequencies over indicated times after transplantation in patients (n = 20 total patients; n = 7 patients with 10 discrete episodes of ACR at any time) based on the presence (solid squares) or absence (open circles) of ACR. ***P < 0.01. (C to E) Serial sections of a TBBx from one patient (P29) who experienced ACR at 1 month after lung transplant (grade 1); donor is HLA-A2+, and recipient is HLA-A2. Immunofluorescence imaging stained for E-cadherin (purple), CD4 (green), HLA-A2 (red), and DAPI (blue). Yellow arrows indicate donor cells, and green arrows indicate recipient cells. “*” identifies a blood vessel (E) H&E stain, and “*” identifies blood vessel (*BV). (F) Frequency of donor origin CD4 (left) and CD8 (right) T cells in the BAL stratified on the basis of the presence or absence of a positive bacterial culture. *P < 0.05.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/33/eaav5581/DC1

    Methods

    Fig. S1. Gating strategy for donor and recipient T cells from BAL samples.

    Fig. S2. Gating strategy for donor and recipient T cells from blood.

    Fig. S3. Lung transplant recipients have low frequencies of CD4+ Tregs in blood and BAL samples.

    Fig. S4. Single-cell RNA sequencing schematic and comparison between patients.

    Fig. S5. Lack of association of donor T cell chimerism in BAL with viral infection.

    Table S1. Patient demographics and transplant characteristics.

    Table S2. Patient demographics for transplant recipients used for CD4+ Treg data.

    Table S3. Summary of scRNA-seq results by a 96-well plate.

    Table S4. Characteristics of the organ donors for control lung samples.

    Table S5. List of antibodies used for flow cytometry and immunofluorescence imaging.

    Table S6. Details of reverse transcriptase primers.

    Table S7. Cell filtration cutoffs for P19.

  • Supplementary Materials

    The PDF file includes:

    • Methods
    • Fig. S1. Gating strategy for donor and recipient T cells from BAL samples.
    • Fig. S2. Gating strategy for donor and recipient T cells from blood.
    • Fig. S3. Lung transplant recipients have low frequencies of CD4+ Tregs in blood and BAL samples.
    • Fig. S4. Single-cell RNA sequencing schematic and comparison between patients.
    • Fig. S5. Lack of association of donor T cell chimerism in BAL with viral infection.
    • Table S1. Patient demographics and transplant characteristics.
    • Table S2. Patient demographics for transplant recipients used for CD4+ Treg data.
    • Table S3. Summary of scRNA-seq results by a 96-well plate.
    • Table S4. Characteristics of the organ donors for control lung samples.
    • Table S5. List of antibodies used for flow cytometry and immunofluorescence imaging.
    • Table S6. Details of reverse transcriptase primers.
    • Table S7. Cell filtration cutoffs for P19.

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