MAdCAM-1 co-stimulation combined with retinoic acid and TGF-β induces blood CD8+ T cells to adopt a gut CD101+ TRM phenotype

Abstract

Resident memory T cells (T_RMs) help control local immune homeostasis and contribute to tissue-protective immune responses. The local cues that guide their differentiation and localization are poorly defined. We demonstrate that mucosal vascular addressin cell adhesion molecule 1, a ligand for the gut-homing receptor α₄β₇ integrin, in the presence of retinoic acid and transforming growth factor-β (TGF-β) provides a co-stimulatory signal that induces blood cluster of differentiation (CD8⁺ T cells to adopt a T_RM-like phenotype. These cells express CD103 (integrin α_E) and CD69, the two major T_RM cell-surface markers, along with CD101. They also express C-C motif chemokine receptors 5 (CCR5) , C-C motif chemokine receptors 9 (CCR9), and α₄β₇, three receptors associated with gut homing. A subset also expresses E-cadherin, a ligand for α_Eβ₇. Fluorescent lifetime imaging indicated an α_Eβ₇ and E-cadherin cis interaction on the plasma membrane. This report advances our understanding of the signals that drive the differentiation of CD8⁺ T cells into resident memory T cells and provides a means to expand these cells in vitro, thereby affording an avenue to generate more effective tissue-specific immunotherapies.

Fig. 1.. MAdCAM-1 costimulation combined with TGF-β and RA promotes a T_RM -like phenotype on CD8⁺ T cells.

(A) Representative flow cytometric dot plots of CD69 (y-axis) and CD103 (x-axis) expression on CD8⁺ T cells following costimulation with anti CD3 plus MAdCAM-1, VCAM or anti CD28, all in the presence of RA and TGF-β. Frequencies of cells in each quadrant are indicated. (B) Frequency of CD69 and CD103 expression on CD8⁺ T cells in 11 independent donors following costimulation as in A, in the presence or absence of RA, as indicated (***:P < 0.01, ****: < 0.0001 ANOVA). (C) Heatmap comparing average RNA expression levels from 4–5 donors for 41 selected genes associated with T_RM cells following CD8⁺ T cells stimulation with anti CD3 + RA (left), anti CD3 + MAdCAM-1 + RA (center), and anti CD3 + MAdCAM-1 + RA + TGF-β (right). Each column represents one donor, each row represents a selected gene. Sideline indicates expected direction of gene modulation on CD8⁺ T_RMs. Asterisks indicate genes presented as violin plots in panels d and e. (D to E) Violin plots of expression of select T_RM associated genes following stimulation as in panel a. anti CD3 + RA (magenta), anti CD3 + MAdCAM-1 + RA (green), anti CD3 + MAdCAM-1 + RA + TGF-β (blue). y-axis indicates relative units (RU). (P values are indicated, on-way ANOVA).

Fig. 2.. Inhibitory receptor expression on CD8⁺ T_RMs generated by MAdCAM-1 costimulation.

(A) t-SNE map showing the cluster distribution of 12 cell surface markers following MAdCAM-1 (blue), VCAM-1 (grey), and CD28 (magenta) costimulation in presence of RA and TGF-β. Each treatment group concatenates data from 4 donors. (B) Overlay of the t-SNE map with the expression levels for CD103 and each inhibitory receptor, as listed. Color based on median fluorescence intensity values. (C) Frequency of CD101 (left), PD1 (middle) and CTLA-4 (right) expression on CD8⁺ T_RMs-like cells from 4 independent donors following costimulation with anti CD3 plus MAdCAM-1, VCAM-1 or anti CD28, all in the presence of RA and TGF-β. (****: P < 0.0001, ***: P < 0.001, *: P < 0.1, one-way ANOVA).

Fig. 3.. MAdCAM-1 requires RA to induce CCR9 expression on CD8⁺ T_RM -like cells.

(A) Representative flow cytometry dot plot of CCR9, CD69 and CD103 coexpression on CD8⁺ T cells following costimulation with anti CD3 combined with either MAdCAM-1, VCAM-1, or anti CD28, all in the presence of RA and TGF-β. CD69 (y-axis), CD103 (x-axis) and CCR9 (green intensity) are shown. Frequencies of cells in each quadrant are indicated. (B) CCR9 frequency on CD69⁺, CD103⁺ CD8⁺ T cells in 10 independent donors following costimulation with anti CD3 combined with either MAdCAM-1, VCAM-1, or anti CD28, and TGF-β in the presence vs absence of RA, as indicated (****: P < 0.0001 ANOVA). (C) RT-qPCR analysis of CCR9 transcription in 6 independent donors following costimulation with anti CD3 combined with either MAdCAM-1, VCAM-1, or anti CD28, all in the presence of TGF-β and RA. Y axis indicates relative expression level (RU). (*: P < 0.5, Kruskal-Wallis, one-way ANOVA). (D) Representative flow cytometry dot plot of CCR5, CD69 and CD103 coexpression on CD8⁺ T cells following costimulation as in panel a. CD69 (y-axis) and CD103 (x-axis) and CCR5 (green intensity) are shown. Frequencies of cells in each quadrant are indicated. (E) CCR5 frequency on CD69⁺, CD103⁺ CD8⁺ T cells in 10 independent donors following costimulation as described for panel b. (F) RT-qPCR analysis of CCR5 gene transcription in 6 independent donors following costimulation as in panel c (Kruskal-Wallis, one-way ANOVA).).

Fig. 4.. α₄β₇ antagonist prevents gut CD8⁺ T_RM -like formation.

Inhibition of CD69 and CD103 induction on CD8⁺ T cells from 3 independent donors by vedolizumab (anti α₄β₇), FIB504 (anti β7), anti MAdCAM-1 or a control IgG mAb, following costimulation with MAdCAM-1 + RA + TGF-β. Y-axis indicates the frequency of CD103⁺ CD69⁺ cells. (**: P < 0.01, *: P < 0.1, one-way ANOVA).

Fig. 5.. Coexpression of E-cadherin and α_Eβ₇ on CD8⁺ T_RM -like cells.

(A) Representative flow cytometric dot plots illustrating the induction of E-cadherin on CD8⁺ CD69⁺ CD103⁺ T cells following costimulation with anti CD3 combined with either MAdCAM-1, VCAM-1 or anti CD28, all in the presence of RA and TGF-β, as indicated. Left panels: CD69 (y-axis), CD103 (x-axis). Right panels: E cadherin (y-axis) expression within the CD69⁺ CD103⁺ population. Frequencies of cells in each quadrant or gate are indicated. (B) Frequency of E-cadherin on T_RM -like cells following costimulation, as in panel a, in 8 independent donors. Y-axis indicates the frequency of E-cadherin⁺ cells within the CD69⁺ CD103⁺ population. (C) E-cadherin mRNA expression (RT-qPCR) following costimulation as in panel b. Average induction in 6 independent donors is shown. y-axis indicates fold induction normalized to a reference gene (Kruskal-Wallis, one-way ANOVA). (D) Confocal images of CD8⁺ T cells following costimulation as in A. Representative cells stained following costimulation as in a with DAPI (blue), anti CD103 (green), anti E-cadherin (red) as indicated. anti CD103 and E-cadherin merged without or with DAPI, and digitally defined regions of colocalization (yellow) with percentages are included.

Fig. 6. E-cadherin and α_Eβ₇ interact in cis on CD8⁺ T_RM -like cells.

FLIM analysis of E-cadherin and CD103 following costimulation in the presence of RA and TGF-β. (A) Confocal (left) and lifetime images (right) from representative CD8⁺ T_RM -like cells. Confocal images show CD103 (green), E-cadherin (red) and merged (yellow), as indicated. Fluorescence lifetime of donor mAb (CD103) on cells in the absence of an acceptor mAb (E-cadherin) (Row A). Donor lifetime in the presence of E-cadherin mAb on a CD103⁺ E-cadherin⁺ cell (Row B) or on a CD103⁺ E-cadherin^neg cell (Row C). Pseudocolor lifetime scale (nanosecond), with cooler color indicating longer lifetime and warmer color indicating shorter lifetime is included. (B) Average fluorescent lifetime of donor mAb alone from 20 ROI (4 cells, 5 ROI/cell) (yellow). Average donor lifetime of CD103 donor mAb in the presence of E-cadherin mAb on a CD103⁺ E-cadherin⁺ (blue) and CD103⁺ E-cadherin^neg (green), both from 80 ROI (16 cells, 5 ROI/cell). Y axis indicates lifetime (ns). Error bars indicate mean w/s.d. (****: P < 0.0001, two-tailed t test).