Tissue-Resident Memory T Cells Mediate Immune Homeostasis in the Human Pancreas through the PD-1/PD-L1 Pathway

Abstract

Non-recirculating tissue-resident memory T cells (TRMs) are the predominant T cell subset in diverse tissue sites, where they mediate protective immune responses in situ. Here, we reveal a role for TRM in maintaining immune homeostasis in the human pancreas through interactions with resident macrophages and the PD-1/PD-L1 inhibitory pathway. Using tissues obtained from organ donors, we identify that pancreas T cells comprise CD8⁺PD-1^hi TRMs, which are phenotypically, functionally, and transcriptionally distinct compared to TRMs in neighboring jejunum and lymph node sites. Pancreas TRMs cluster with resident macrophages throughout the exocrine areas; TRM effector functions are enhanced by macrophage-derived co-stimulation and attenuated by the PD-1/PD-L1 pathways. Conversely, in samples from chronic pancreatitis, TRMs exhibit reduced PD-1 expression and reduced interactions with macrophages. These findings suggest important roles for PD-1 and TRM-macrophage interactions in controlling tissue homeostasis and immune dysfunctions underlying inflammatory disease, with important implications for PD-1-based immunotherapies.

Keywords: PD-1; chronic pancreatitis; macrophage; memory T cells; mucosal immunity; pancreas; tissue immunity.

Figure 1.. Localization and Expression of Key Tissue-Residency Markers on T Cells in Human Pancreas

(A) Representative qmIF composite image of a pancreas section stained with antibodies specific for CD3 (purple), the ductal marker CK19 (green), DAPI nuclear counterstain (gray), and the neuroendocrine marker chromogranin (white) are shown (left) adjacent to a representative single color CD3 image (middle). Acinar, ductal, and endocrine areas were defined based on CK19 and chromogranin staining. White bar, 100 µm for scale. Right: densities of CD3⁺ T cells were quantified in the three regions of pancreas using inForm software. Plots show mean ± SEM from 13 donors. (B) T cells were analyzed in cell suspensions of pancreas (Panc), jejunum (Jej), pancreas-draining lymph node (PLN), and mesenteric lymph node (MLN). Shown are representative (left) and the compiled (right) CD4 and CD8 T cell frequencies (gated on DAPI^lo CD45⁺CD3⁺ cells) from the four tissue sites. Bars indicate comparisons for CD8⁺ T cells. (C) Expression of CD69 in conjunction with TRM signature markers CD103, CD49a, and PD-1 on CD8⁺ TEM cells (CD45RA⁻CCR7⁻) subsets isolated from indicated sites shown as representative flow cytometry plots (left) with the compiled frequencies ±SEM of the indicated subsets from three to eight donors (right). Bars indicate comparisons of the CD69⁺CD103⁺ (top), CD69⁺CD49a⁺ (middle), and CD69⁺PD-1^hi (bottom) subsets. (D) Expression of intracellular granzyme B (GZMB) in CD8⁺CD69⁺TEM cells isolated from pancreas, jejunum, and PLN shown as representative flow cytometry plots (left), and compiled frequencies ± SEM of GZMB⁺ cells from three to six donors for each tissue (right). Bars indicate comparisons of the GZMB⁺ frequencies within the indicated subsets. **p < 0.001 as calculated by two-way ANOVA with Dunnett’s multiple comparisons test. See also Figure S1.

Figure 2.. TRMs in the Pancreas Express a Tissue-Specific Transcriptional Profile Associated with Enhanced T Cell Function and Mitochondrial Mass

CD8⁺ TRMs (CD8⁺CD69⁺ TEM) were sorted from pancreas, PLN, and jejunum along with blood (BL) CD8⁺CD69⁻ TEM cells for whole-transcriptome profiling by RNA-seq. (A) Principal-component analysis (PCA) of gene expression profiles of tissue CD8⁺ TRMs compared to blood CD8⁺CD69⁻ TEM cells. (B) Gene set enrichment analysis (GSEA) comparing expression of genes in pancreas TRMs versus blood CD8⁺ TEM cells to a core human TRM gene signature (Kumar et al., 2017). (C) Biplots of all genes showing log FC of pancreas/blood versus log FC of jejunum/blood (top) and log FC of pancreas/PLN versus log FC of pancreas/jejunum (bottom). Significantly differentially expressed genes (DEGs) for each comparison were identified using DeSeq and functionally classified and are indicated as colored dots based on gene pathway. (D) Heatmaps showing representative DEG from annotated gene sets T cell activation, proliferation, migration (left) and mitochondrion (right) which significantly overlap with DEGs in pancreas versus PLN and pancreas versus jejunum. (E) Determination of mitochondrial mass using MitoTracker green staining in verapamil-treated CD8⁺ TRMs from matched donor sites pancreas, jejunum, and PLN. Representative histogram of MitoTracker green signal in matched sites from one donor is shown (top) with the compiled frequencies ±SEM from three donors for each tissue (bottom). (F) Sorted CD8⁺CD69⁺ TEM subsets isolated from pancreas, jejunum, PLN, and sorted blood CD8⁺CD69⁻ TEM cells and naive CD8⁺ T cells were stimulated with PMA and ionomycin, and intracellular staining was used to assess IFN-γ, IL-2, and TNF-α production. Profiles of IFN-γ and IL-2 are shown in representative flow cytometry plots for the indicated tissues (left). Graphs of compiled frequencies of IFN-γ -producing cells and the polyfunctionality index (see STAR Methods) ±SEM from three to six donors for each tissue (right). **p < 0.01 as calculated by one-way ANOVA with Dunnett’s multiple comparisons test. See also Figure S2.

Figure 3.. Pancreas TRMs Exhibit Tissue-Specific Clonal Expansion with Phenotypic Features of Previous Replication

TCR sequences of CD8⁺ TRMs from pancreas, jejunum, and PLN were extracted from RNA-seq data using MIXR (see STAR Methods). (A) Representative clone tracking analysis showing the distribution and degree of expansion of PLN clonotypes across tissues. Each line represents a tracked clone present in PLN (far left, arrow) and at least one of the other tissues. Line thickness indicates degree of clonal expansion at the indicated site and line color indicates the tissue site in which that clone exhibits the greatest degree of clonal expansion. (B) The percentage of the total TCR sequences (±SEM from three to five donors for each tissue) detected in pancreas and jejunum TRM that are either shared or not shared with PLN clonotypes is shown (top). The diversity of the TCR repertoires in TRMs isolated from indicated tissues is depicted as the Chao1 index (bottom). (C) The percent contribution of expanded clonotypes from TRMs isolated from pancreas (top) and jejunum (bottom) to the total TCR repertoire extracted from pancreas, jejunum, and PLN (±SEM from three to five donors for each tissue). **p < 0.01, *p < 0.05 as calculated by one-way ANOVA with Dunnett’s multiple comparisons test. (D) Coordinate expression of CD127 and CD28 by CD8⁺CD69⁺ TEM cells isolated from pancreas, jejunum, PLN, and MLN shown as representative flow cytometry plots (left), with the compiled frequencies (±SEM) of indicated subsets from 9 to 20 donors for each tissue (right). **p < 0.01 as calculated by two-way ANOVA with Sidak’s multiple comparisons test. Bars indicate comparisons of the CD127⁺CD28⁻ subset. See also Figure S3.

Figure 4.. Localization and Phenotype of Pancreas Macrophages

(A) Light scatter—forward scatter (FSC) and side scatter (SSC)—profile of DAPI^loCD45⁺ cells from a pancreas cell suspension after dissociation in a Ricordi chamber (left). The proportion of these cells expressing the myeloid lineage markers CD14 and CD64 is shown in a representative flow cytometry plot (center) with the compiled percentages of the indicated CD14⁺CD64⁺ subset (right) (± SEM from 12 to 16 donors for each tissue). (B and C) Expression of tissue macrophage markers CD163 and CD206 (B), as well as MHC class II and CD86 (C), on the DAPI^lo CD45⁺CD64⁺CD14⁺ myeloid cells isolated from pancreas, jejunum, PLN, and spleen. Shown are representative flow cytometry plots (left) with the compiled percentages of the indicated subsets (right) (±SEM from 12 to 16 donors for each tissue). **p < 0.01 as calculated by one-way ANOVA with Dunnett’s multiple comparisons test. (D) Density of pancreas macrophages in endocrine and exocrine pancreas. Shown is a representative qmIF image with the single-marker CD163 (left) and the corresponding composite image (right) of CD163 (yellow), the ductal marker CK19 (green), and the neuroendocrine marker chromogranin (white). Acinar, ductal, and endocrine areas were defined using inForm software, and the densities of CD163⁺ macrophages were quantified in the indicated pancreatic tissue areas (right) (± SEM from 13 donors). **p < 0.01 as calculated by one-way ANOVA with Sidak’s multiple comparisons test. White bar, 100 µm for scale. See also Figure S4.

Figure 5.. TRMs Cluster with Macrophages in the Exocrine Pancreas

(A) Composite image of human pancreas acquired using the Vectra platform (left) for quantitative multiplex immunofluorescence (qmIF) showing the ductal marker CK19 (green), the neuroendocrine marker chromogranin (white), and the macrophage marker CD163 (yellow). CD3⁺CD103⁺ T cells are shown in purple, and CD3⁺CD103⁻ T cells are shown in blue. A map of the ductal, neuroendocrine, and T cells identified using inForm software is shown (right). (B) PCFs based on qmIF images were calculated between each pair of cell types. Each line represents the median PCF across samples, and shaded regions are 95% confidence intervals of the median (left). The area under the curve (AUC) for PCFs were compared among three cell pairs: (1) macrophages versus any cell (gray), (2) macrophages versus CD103⁻ T cells (blue), and (3) macrophages versus CD103⁺ T cells (purple). AUC has units of microns, and a random Poisson-distributed sample would have an AUC of 50. ***p < 0.001, Mann-Whitney U test (right). See also Figure S5.

Figure 6.. Functional Regulation of Pancreas TRMs by CD58 and PD-L1 Pathways

Sorted pancreas TRMs (CD8⁺CD69⁺ TEM cells) were stimulated with monomeric TCR-activating anti-CD3 antibody (OKT3) in the presence or absence of sorted pancreas macrophages (MF; CD14⁺CD64⁺ CD163⁺) (1:1 ratio) or with microbeads coated with anti-CD3 and exogenous co-stimulation (activating anti-CD2 and anti-CD28 antibodies). (A) Cytokine production (IFN-γ and IL-2) with the indicated stimulation conditions is shown in representative flow cytometry plots (left) with compiled data for each cytokine and the cytokine polyfunctionality index (right) (±SEM from three to five donors). **p < 0.01, as calculated by one-way ANOVA with Dunnett’s multiple comparisons test. (B and C) Representative histograms of PD-L1 (B) and CD58 (C) expression on pancreatic macrophages (CD45⁺CD14⁺CD163⁺, red), non-myeloid immune cells (CD45⁺ CD14⁻, blue), and CD45⁻ cells (gray) with graphs of compiled frequencies (±SEM from four to seven donors) expressing the indicated marker (right). **p < 0.01 macrophages versus both other cells types as calculated by one-way ANOVA with Dunnett’s multiple comparisons test. (D) Pancreas TRMs in macrophage co-cultures were stimulated with anti-CD3 in the presence or absence of PD1 blocking antibody (nivolumab) or CD58 blocking antibody followed by intracellular cytokine staining. Shown are flow cytometry profiles of TRM IFN-γ and IL-2 production (top) with compiled data showing the frequencies of TRMs producing each cytokine and the cytokine polyfunctionality index (bottom) (±SEM from four donors). **p < 0.01, *p < 0.05 as calculated by one-way ANOVA with Dunnett’s multiple comparisons test. See also Figure S6.

Figure 7.. Alterations in Pancreatic Immune Cell Composition and T Cell Regulation in Patients with Chronic Pancreatitis

Samples of unfractionated pancreatic cell suspensions were obtained from research subjects undergoing total pancreatectomy with auto-islet transplantation. (A) Pancreas immune cells from patients with CP and age-matched organ donor controls without evidence of pancreatic disease. Shown are representative (left) and compiled frequencies (right, ±SEM) of total T cells (first column, gated on DAPI^loCD45⁺CD3⁺ cells) and macrophages (second column, gated on DAPI^lo CD45⁺Lin(CD19/20/56/15/16/66b)⁻CD14⁺CD64⁺ cells). (B) Graphs of compiled data show the T cell to macrophage ratio (top) and frequencies of CD8⁺ TRMs (bottom, gated on DAPI^loCD45⁺CD3⁺CD8⁺CD45RA⁻ CCR7⁻CD69⁺) in control and CP pancreata. Data shown are from 19 controls and 8 CP patients. (C) Representative micrographs (203 magnification) of single-color immunohistochemistry showing CD8 (top) and CD163 (bottom) staining in control (left) and CP pancreas (right). White bar, 100 µm for scale. (D) Expression of PD-1 on CD8⁺ TRMs (left) and PD-L1 on macrophages (right) from pancreas of control (blue) and CP patients (red) shown in representative histograms (top), with graphs of compiled mean fluorescence intensities (MFIs). (E) Expression of T-bet in CD8⁺ TRMs is shown from pancreas of controls (blue) and CP patients (red) in representative histograms (left) and graphs of compiled MFIs (±SEM from five controls and five CP patients). **p < 0.01, *p < 0.05 as calculated by unpaired t test. (F) Linear regression showing the relationship between PD-1 and T-bet MFI on pancreas CD8⁺ TRMs of CP patients and controls with 95% confidence bands of the best-fit line. See also Figure S7.