An evolving autoimmune microenvironment regulates the quality of effector T cell restimulation and function

Abstract

Defining the processes of autoimmune attack of tissues is important for inhibiting continued tissue destruction. In type 1 diabetes, it is not known how cytotoxic effector T cell responses evolve over time in the pancreatic islets targeted for destruction. We used two-photon microscopy of live, intact, individual islets to investigate how progression of islet infiltration altered the behavior of infiltrating islet-specific CD8(+) T cells. During early-islet infiltration, T-cell interactions with CD11c(+) antigen-presenting cells (APCs) were stable and real-time imaging of T cell receptor (TCR) clustering provided evidence of TCR recognition in these stable contacts. Early T cell-APC encounters supported production of IFN-γ by T effectors, and T cells at this stage also killed islet APCs. At later stages of infiltration, T-cell motility accelerated, and cytokine production was lost despite the presence of higher numbers of infiltrating APCs that were able to trigger T-cell signaling in vitro. Using timed introduction of effector T cells, we demonstrate that elements of the autoimmune-tissue microenvironment control the dynamics of autoantigen recognition by T cells and their resulting pathogenic effector functions.

Keywords: 2-photon microscopy; T-lymphocyte reactivation; autoimmunity.

Fig. 1.

T cells arrest during early infiltration and regain motility with increased infiltration. CD2-dsRed.OT-I CD8⁺ T cells or 10% CD2-dsRed.OT-I CD8⁺ T cells plus 90% OT-I CD8⁺ T cells were transferred into RIP-mOva or RIP-mOva.CD11c-YFP recipient mice. Islets were isolated 4–7 d following T-cell transfer, immobilized in low-melting agarose, and imaged by time-lapse two-photon microscopy. (A) Representative maximum intensity projection images of T-cell (red) motility in islets with light, mid, and heavy T-cell infiltration. All infiltrating OT-I T cells are visualized in the islet with light infiltration whereas only 10% of infiltrating OT-I T cells are visualized in the islets with mid and heavy infiltration. White tracks show the path of T-cell motility over 10 min. The dashed gray line indicates the islet border. (Scale bars: 30 μm.) (B) Ten randomly selected T-cell tracks from the islets shown in A. Scale shown in μm. (C) Mean squared displacement of T cells over 10 min time. Analysis includes all cells tracked in A. Error bars = SEM. (D and E) Quantification of T-cell motility. Each dot represents the average of all tracked T cells in one islet, and the red bar represents the mean of all islets. Data were combined from 82 islets in 13 experiments. Statistical analyses were done using a 1-way ANOVA Kruskal–Wallis test with Dunn’s Multiple Comparison Test. *P < 0.05, **P < 0.001, ***P < 0.0001. (D) Average speed of T-cell crawling in infiltrated islets. (E) Arrest coefficient of T cells in infiltrated islets.

Fig. 2.

T cell–APC interactions convert from sustain to transient with increased infiltration. Isolated islets were imaged by time-lapse two-photon microscopy as described for Fig. 1. *P < 0.05, **P < 0.001, ***P < 0.0001. (A) Representative time-lapse imaging of sustained T-cell interactions in a lightly infiltrated islet. Filled arrowheads indicate sustained interactions of a T cell with a CD11c⁺ cell, and open arrowheads indicate sustained interactions of a T cell with an unlabeled cell. (Scale bar: 30 μm.) Time-lapse area shown represents 90 μm (x) × 90 μm (y) × 15 μm (z). Time stamp, min:sec. (B–E) Quantification of T cell–CD11c+ cell interactions. Data were combined from nine experiments. (B) Percentage of T cells that contacted a CD11c⁺ cells for at least 1 min. (C) Percentage of T cells that maintain T cell–CD11c⁺ cell interactions for at least 10 min. (B and C) Statistical analyses were done using a Chi Square Test. (D and E) Duration of the longest T cell–CD11c+ cell interaction per interacting T cell. (E) Each dot represents one T cell, and the red bar represents the median. Statistical analyses were done using a 1-way ANOVA Kruskal–Wallis test with Dunn’s Multiple Comparison Test.

Fig. 3.

CD11c⁺ APCs in heavily infiltrated islets maintain the ability to trigger T cells. OT-I T cells were transferred into RIP-mOva recipients. Islets were isolated at different stages of infiltration: (−) no transfer control, (d4.5) early, (d7) late. CD11c⁺MHCII^hiDAPI^– APCs sorted from the spleen or islets were incubated with in vitro-activated OT-I T cells labeled with Fura-2AM. Splenic APCs were antigen-pulsed. T cell–APC interactions and Fura fluorescence were imaged by time-lapse widefield microscopy. Data combined from two experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (A) Representative examples of calcium fluxes read out by Fura-2AM 340 nm/380 nm ratios. (B) Quantification of T cell–APC contacts and calcium-flux strength in T cells interacting with APCs. Statistics: Chi Square Test.

Fig. 4.

Stable T cell–APC interactions induce TCR signaling and APC killing in the islets. (A) TCR central supramolecular activating complex (cSMAC) formation and internalization during interactions with CD11c⁺ cells in the islets. OTI-TCR-GFP T cells were transferred into RIP-mOva.CD11c-mCherry recipients, and isolated islets were imaged by two-photon microscopy. TCR density is on a pseudocolor scale. Arrows indicate location of cSMAC, with internalized TCR visible in the last frame. Representative of two experiments. (B and C) APC killing by T cells in the islets. CD2-dsRed.OT-I T cells were transferred into RIP-mOva.CD11c-YFP recipients, and islets were imaged by two-photon microscopy. (B) Representative time-lapse of T cell-induced APC killing in a lightly infiltrated islet. Arrows indicate APC killing. Time-lapse represents 105 μm (x) × 105 μm (y) × 15 μm (z). Time stamp, min:sec. (C) Quantification of APC killing. Data combined from 10 experiments. **P < 0.01 by 1-way ANOVA Kruskal–Wallis test with Dunn’s Multiple Comparison Test.

Fig. 5.

T-cell dynamics and effector cytokine production are determined by the state of islet infiltration. (A) Experimental set-up for (B–D). (B) Induction of IFN-γ production in the islets is associated with T-cell arrest. Representative plots of in vivo IFN-γ production by intracellular cytokine staining. (C) Quantification of data shown in B. Data combined from six mice in three experiments. Statistics: 1-way ANOVA Kruskal–Wallis test with Dunn’s Multiple Comparison Test. *P < 0.05. (D) Environmental factors prevent T-cell arrest in heavily infiltrated islets. Quantification of T-cell arrest coefficients analyzed by time-lapse two-photon imaging of T cells in early (d5) or late (d7) infiltrated islets. (E) CD25 expression is lost as T cells become more motile. Four animals per group combined from two experiments. **P < 0.01 by 1-way ANOVA Kruskal–Wallis test with Dunn’s Multiple Comparison Test. (F–H) CD11c⁺CX3CR1^low APCs are recruited to the islets with infiltration. APC populations analyzed on a per-islet basis by two-photon microscopy at varying degrees of infiltration in RIP-mOva.CX3CR1-GFP.CD11c-YFP mice. Each dot represents one islet. (F) Uninfiltrated islets were analyzed in mice without T-cell transfer. **P < 0.01 by two-tailed t test. (G) Analysis of CX3CR1^low APCs in infiltrated islets. CX3CR1^low APCs correlate with T-cell infiltration. (H) Average cell count per islet of CX3CR1^low versus CX3CR1^high CD11c+ APCs with and without islet infiltration. ***P < 0.0001 by 2-way ANOVA with Tukey’s multiple comparison test.