A local regulatory T cell feedback circuit maintains immune homeostasis by pruning self-activated T cells

Abstract

A fraction of mature T cells can be activated by peripheral self-antigens, potentially eliciting host autoimmunity. We investigated homeostatic control of self-activated T cells within unperturbed tissue environments by combining high-resolution multiplexed and volumetric imaging with computational modeling. In lymph nodes, self-activated T cells produced interleukin (IL)-2, which enhanced local regulatory T cell (Treg) proliferation and inhibitory functionality. The resulting micro-domains reciprocally constrained inputs required for damaging effector responses, including CD28 co-stimulation and IL-2 signaling, constituting a negative feedback circuit. Due to these local constraints, self-activated T cells underwent transient clonal expansion, followed by rapid death ("pruning"). Computational simulations and experimental manipulations revealed the feedback machinery's quantitative limits: modest reductions in Treg micro-domain density or functionality produced non-linear breakdowns in control, enabling self-activated T cells to subvert pruning. This fine-tuned, paracrine feedback process not only enforces immune homeostasis but also establishes a sharp boundary between autoimmune and host-protective T cell responses.

Keywords: CTLA-4; IL-2; IL-2Rα; apoptosis; autoimmunity; computational modeling; feedback control; immune homeostasis; quantitative tissue imaging; regulatory T cells.

Figure 1. Tregs selectively accumulate around self-activated T cells, forming micro-domains.

(A) Left side: PD-1 expression variation among paracortical CD4⁺ T cells. Dashed yellow line: popliteal LN paracortex. White box: region of interest (ROI). Right side: Magnified ROI. XZ optical slices highlight cell of interest (white arrow). Scale bars = 200 μm (left) and 20 μm (right) (B) In situ quantification of A. TRAP = anti-CD62L Ab + FTY720. n = 3-4 animals. Data are from 2 independent experiments. (C) Spatial density function of Tregs shown in A. White dots: PD-1⁺ CD4⁺ T cells within the paracortex. Scale bar = 200 μm. (D) Local Treg densities surrounding PD-1⁺ CD4⁺ T cells or randomly sampled PD-1⁻ CD4⁺ T cells. Solid lines: local regressions determined by the LOESS method. Error bars: 95% confidence intervals. Data are representative of 3 independent experiments.

Figure 2. Paracrine IL-2 signaling initiates local Treg feedback

(A) Multiplexed imaging of Treg micro-domains in inguinal LNs. Left panel: Treg micro-domain. Arrows: PD-1⁺ CD4⁺ T cell. Scale bar = 20 μm. Right panel: Image gallery. Inner and outer zones (yellow dashed lines) depict Tregs inside and outside of the micro-domain, respectively. White dashed circle: PD-1⁺ CD4⁺ T cell. (B) Unsupervised hierarchical clustering of paracortical Tregs (k = 3477 cells) during homeostasis. Representative of 3 independent experiments (C) Tregs from each cluster in B residing within 40 μm of PD-1⁺ CD4⁺ T cells (k = 260 cells) or randomly sampled PD-1⁻ CD4⁺ T cells (k = 260 cells). ***p < 0.0001 determined using a two-way ANOVA with the Tukey correction (D) Log2 fold-change in local Treg Foxp3, CTLA-4, and IL-2Rα expression 24h post-IL2 blocking Abs. Dashed red line: isotype control condition. Individual cells pooled from n = 3 animals. Data are mean ± 95% confidence intervals derived using non-parametric bootstrapping. ***p < 0.0001 determined using a one-tailed Student’s T test (E) pSTAT5 signal in Foxp3DTR^+/+ animals treated with DTX for 7h. Inner dashed circle: PD-1⁺ CD4⁺ T cell. Outer dashed circle: Treg micro-domain. Scale bar = 20 μm. (F) PDFs of pSTAT5⁺ Tregs in the popliteal LN paracortex 7h after injecting PBS (left) or DTX (right). White dots: pSTAT5⁺ CD4⁺ T cells. Scale bar = 250 μm (G) pSTAT5⁺ CD4⁺ T cell (left) or pSTAT5⁺ Treg (right) frequencies in the popliteal LN paracortex. Data are mean ± SEM. Each dot represents an individual mouse pooled from two independent experiments. p values determined using an unpaired, two-tailed t test. (H) Single-cell PD-1 expression in paracortical Foxp3⁻ CD4⁺ IL-2 responders (pSTAT5⁺) or non-responders (pSTAT5⁻) 7h post-DTX. n = 4 animals. Data are from 2 independent experiments.

Figure 3. Self-activated T cells proliferate despite local IL-2 constraints but are rapidly pruned from the host.

(A) Top panel: experimental schematic. Bottom panel: absolute number of cells per gastric LN. Each dot represents a LN from a single mouse. Local regressions with 95% confidence intervals are shown for each cell type over time. Gastric LNs from n = 3-8 mice were imaged at each time point. Data are pooled from 2-3 independent experiments (B) C_e3D imaging of the gastric LN at 72h post-transfer. Insets: magnified ROIs (white dashed boxes). Scale bars = 100 μm and 20 μm (Inset) (C) In situ quantification of TxA23 cell proliferation at 96h post-transfer without FTY-720 and anti-CD62L antibodies. A Gaussian mixture model (GMM) was fit to the CellTracker DeepRed distribution. k = 552 TxA23 cells pooled from gastric LNs of n = 4 animals. Data are from two independent experiments. Inset: fraction of TxA23 cells within each mixture component. Data are Mean ± 95% confidence intervals (D) Ki67 expression in paracortical CD4⁺ T cells. Left side: PD-1⁺ CD4⁺ T cells surrounded by high densities of Tregs. Right side: Magnified image gallery of the ROI (white dashed box). Arrows: PD-1⁺ CD4⁺ T cells enriched in Ki67. Scale bar = 40 μm and 20 μm (ROI). (E) Ki67 versus PD-1 expression in paracortical CD4⁺ T cells of GF C57BL/6 nice. Data are representative of 3 independent experiments. (F) Enriched active caspase 3 expression in a PD-1⁺ CD4⁺ T cell. Inset: magnified ROI (white dashed box). Scale bars = 20 μm and 5 μm (inset). (G) IL-2:S4B6-1 innunoconplex schematic. (H) In situ quantification of active caspase 3⁺ cells. Data are mean ± SEM. Each dot represents an individual nouse pooled from 2 independent experiments. p values determined using a one-way ANOVA with the Tukey correction.

Figure 4. Treg micro-domain formation is part of the IL-2-driven feedback process

(A) 1x10⁵ TxA23 cells and WT cells were co-transferred into BALB/c recipients. Local densities of pSTAT5⁺ Tregs (λ_pSTAT5+Treg - top panel) and total Tregs (λ_Treg – bottom panel) surrounding individual TxA23 cells (nagenta) or WT cells (green). Local regressions with 95% confidence intervals determined by the LOESS method. k = 25-150 cells pooled from gastric LN sections of n = 4-6 animals at each tine point. Data are from 2 independent experiments. (B) pSTAT5 signal in Tregs surrounding a WT cell (top panel) or TxA23 cell (bottom panel) in the sane gastric LN 24h post-transfer. Scale bar = 20 μm. (C) Adoptive transfers performed as in Figure 3A while co-injecting IL-2 blocking Abs or isotype controls. Gastric LNs harvested 24h post-transfer. Scale bar = 20 μm (D) Maximum deviation in λ_Treg (Δ λ_Treg) between TxA23 vs. WT cells 24h after injecting IL-2 blocking Abs or isotype controls (E) Δ λ_Treg between PD1⁺ CD4⁺ T cells vs. randomly sampled PD-1⁻ CD4⁺ T cells 24h after injecting C57BL/6 nice with IL-2 blocking Abs or isotype controls. For D and E, 95% confidence intervals were derived by non-parametric bootstrapping. Data are from 2 independent experiments.

Figure 5. Nascent micro-domains are a product of localized Treg proliferation

(A) Perspective plot illustrating the local Treg density weighted to local Treg Ki67 expression within a popliteal LN section. Dashed white lines: B cell follicles (B) Ki67 expression in individual Tregs as a function of intercellular distance. The experimentally derived probability distribution (black line) was compared to a random permutation null model. Upper and lower pointwise envelopes (shaded purple regions) are shown for 99 Monte Carlo simulations. Dashed purple line represents the average of the simulations. p = 0.02 at a distance of 5 μm using a two-sided Monte Carlo test. Data is representative of 3 independent experiments. (C) Fluorescent protein recombination frequencies in Tregs 14 days following tamoxifen. Each dot represents an individual animal. (D) Clonal Treg clusters (dashed white circles) in Foxp3CreERT2^+/−-Confetti^fl/− mice. White traces: individual Tregs of interest expressing Foxp3. Scale bars = 20 μm. (E) Local density of Tregs expressing the same FP within 40 μm of one another. Observed FP frequencies were permuted at random across paracortical Treg positions. Points that reside on the dashed black line (slope = 1) are explained by chance. LNs from 4 representative mice were quantified and pooled from 2 independent experiments.

Figure 6. A computational model predicts that modest reductions in micro-domain size or functionality enable self-activated T cells to respond to IL-2.

(A) Schematic illustrating multiscale model dynamics (B) Distribution of pSTAT5^CD4-max values from 20000 dynamic simulations. Inset: log10 scale. Dashed red line: discrete threshold for parameter configurations within the top 5% of the distribution (C) Two-dimensional visualization of sampled parameter space using t-SNE. Each dot represents a parameter configuration. Colored dots represent configurations with pSTAT5^CD4-max values within the top 5% of the distribution (D) Heatmap illustrating individual parameters (columns) and their standard scores (Z-score) within each configuration (rows) from the top 5% of the pSTAT5^CD4-max distribution. (E) Global variable importance (GVI) of dynamical model parameters (y-axis) fitted by the RFML regression model using “combined data” (see Figure S5B). (F) and (G) Percentage of active, inactive, and susceptible configurations in the parameter space following indicated perturbations. (H) Susceptible configurations were pooled from each perturbation in G and visualized within parameter space (red dots). (I) GVI of the RFML classification model comparing susceptible and active configurations. (J) Pearson correlation matrix of individual parameters in susceptible configurations.

Figure 7. Modest reductions in Treg micro-domain size or functionality promote non-linear breakdowns in control

(A) Schematic depicting inducible systems used to manipulate Treg parameters in vivo. (B) Log2 fold-changes in paracortical pSTAT5⁺ CD4⁺ T cells or pSTAT5⁺ Treg frequencies. Individual dots represent individual mice. p values determined using Welch’s two-sided t test comparing the treated animals to their respective controls (dashed red line) (C) PD-1 expression in IL-2 responders (pSTAT5⁺) vs. non-responders (pSTAT5⁻). Single-cell distributions pooled from n = 3-5 animals (D) 60% WT + 40% Foxp3-DTR^+/+ chimeras were injected with DTX. Micrographs highlight a pSTAT5⁺ PD-1⁺ CD4⁺ T cell (dashed white circle) surrounded by a low density of Tregs. Scale bars = 10 μm. (E) Foxp3CreERT2^+/+-CTLA-4^+/fl were injected with tamoxifen. Micrographs highlight a PD-1⁺ CD4⁺ T cell exhibiting high pSTAT5 signal (dashed white circle), despite high local Treg density. Scale bars = 10 μm. (F) Log2 fold-change in active caspase 3⁺ CD4⁺ T cells in the paracortex. Data were normalized to respective vehicle controls (dashed red line). Each dot represents an individual mouse. p values determined using a one-way ANOVA with the Tukey correction. (G) pSTAT5 signal in 5C.C7 cells activated by the partial agonist (T102S) or the agonist (MCC_(88-103)) at 48h post-injection. Dashed circles: 5C.C7 cells of interest. Scale bar = 10 μm. (H) Quantification of G. Each dot represents an individual mouse from two independent experiments. Data are mean ± SEM. p value determined using a two-tailed, Student’s t test.