Tissue-infiltrating alloreactive T cells require Id3 to deflect PD-1-mediated immune suppression during GVHD

Abstract

Persisting alloreactive donor T cells in target tissues are a determinant of graft-versus-host disease (GVHD), but the transcriptional regulators that control the persistence and function of tissue-infiltrating T cells remain elusive. We demonstrate here that Id3, a DNA-binding inhibitor, is critical for sustaining T-cell responses in GVHD target tissues in mice, including the liver and intestine. Id3 loss results in aberrantly expressed PD-1 in polyfunctional T helper 1 (Th1) cells, decreased tissue-infiltrating PD-1+ polyfunctional Th1 cell numbers, impaired maintenance of liver TCF-1+ progenitor-like T cells, and inhibition of GVHD. PD-1 blockade restores the capacity of Id3-ablated donor T cells to mediate GVHD. Single-cell RNA-sequencing analysis revealed that Id3 loss leads to significantly decreased CD28- and PI3K/AKT-signaling activity in tissue-infiltrating polyfunctional Th1 cells, an indicator of active PD-1/PD-L1 effects. Id3 is also required for protecting CD8+ T cells from the PD-1 pathway-mediated suppression during GVHD. Genome-wide RNA-sequencing analysis reveals that Id3 represses transcription factors (e.g., Nfatc2, Fos, Jun, Ets1, and Prdm1) that are critical for PD-1 transcription, exuberant effector differentiation, and interferon responses and dysfunction of activated T cells. Id3 achieves these effects by restraining the chromatin accessibility for these transcription factors. Id3 ablation in donor T cells preserved their graft vs tumor effects in mice undergoing allogeneic hematopoietic stem cell transplantation. Furthermore, CRISPR/Cas9 knockout of ID3 in human CD19-directed chimeric antigen receptor T cells retained their antitumor activity in NOD/SCID/IL2Rg-/- mice early after administration. These findings identify that ID3 is an important target to reduce GVHD, and the gene-editing program of ID3 may have broad implications in T-cell-based immunotherapy.

Graphical abstract

Figure 1.

Id3 maintains the GVHD-inducing capacity of alloreactive T cells. Balb/c mice were subjected to total body irradiation (4.5 Gy on day −1 and 4 Gy on day 0) followed by infusion of 5 × 10⁶ B6 × B6/SJL F1 mouse (CD45.1⁺CD45.2⁺) TCD bone marrow (TCD-BM) alone or together with 5 × 10⁵ B6 (CD45.1^-CD45.2⁺) naïve WT or Id3-cKO CD4⁺ T cells. (A) Survival of Balb/c recipients. (B) Cutaneous GVHD in Balb/c mice receiving WT and Id3-cKO CD4⁺ T cells, day 22 after transplantation in at least 8 mice per group. (C) Intestine from Balb/c recipients were harvested from day 14 to 22, sectioned, and stained with hematoxylin and eosin. Slides were scanned with Leica Aperio VERSA slide scanner and visualized with Aperio ImageScope (20×). N = 8. (D-G) Balb/c recipients were euthanized at the indicated time points after HSCT. Tissues were harvested for the analysis of donor T-cell immune response using flow cytometry. Id3-cKO donor CD4⁺ T cells exhibited normal cytokine-producing capacity represented by the similar percentage (D) and number (E) of IFN-γ⁺ cells in the spleen, mesenteric lymph node (mLN), and liver. (F) Graphs show the number of intestine-infiltrated donor CD4⁺ T cells and those with IFN-γ–producing capacity recovered in Balb/c mice that underwent transplantation with Id3-cKO T cells. (G) Graphs show the frequency of donor T cells expressing α4β7, CCR5, and CCR9. ∗P < .05; ∗∗∗P < .001; Student t test was used for 2-group comparison. At least 4 mice per group were analyzed.

Figure 2.

Id3 represses genes associated with T-cell effector differentiation and inhibitory signaling. WT and Id3-cKO naïve CD4⁺ T cells were activated and cultured under Th1 polarization condition. Cells were harvested 24 hours (TCR primed) and 96 hours after activation. 96 hour-cultured cells were sorted into CD62L^hi and CD62L^lo (Th1 cells) populations before library preparation. (A) Gene set enrichment analysis using Hallmark gene set showed significant enrichment of tumor necrosis factor α signaling via NF-κb, IFN-γ response, IL-2–STAT5 signaling, IL-6–STAT3 signaling, allograft rejection, IFN-α response, and apoptosis comparing transcriptome from 4-day cultured Id3-cKO Th1-like cells with WT. (B) Id3-regulated gene signature in TCR-primed CD4⁺ T cells and effector Th1 CD4⁺ T cells. (C-G) Heat maps demonstrate 5 categories of differential expressed genes: TFs critical for effector proliferation and differentiation (C); cytokines that distinguish Th1 from Th2 and Th17 cells (D); cell survival and death molecules (E); costimulatory molecules important for GVHD T-cell proliferation and survival (F); and inhibitory receptors (G). Data were collected from 3 independent experiments. (H) Flow cytometry analysis shows that loss of Id3 leads to increased expression of PD-1 (H, I) and PD-L1 (J) in Th1 cells. ∗∗∗P < .001. Experiments were performed >5 times.

Figure 3.

Id3 deficiency leads to PD-1 upregulation on polyfunctional Th1 cells. (A-C) Approximately 6 to 8 days after allo-HSCT, cells were isolated from the spleen and liver of Balb/c recipients of donor B6 naïve T cells as described in Figure 1, and donor T cells were analyzed for effector function. Plots and graphs show the percentage of IFN-γ⁺ GM-CSF⁺ polyfunctional cells (A-B) and the number of Id3-cKO IFN-γ⁺GM-CSF⁺ T cells in the spleen and liver (C). (D-E) Plots and graphs show the frequency of lamina-propria lymphocyte (LPL) IFN-γ⁺GM-CSF⁺ cells in the intestine of Balb/c recipients 17 days after HSCT. (F-H) The IFN-γ⁺GM-CSF⁺ polyfunctional population was gated for the analysis of inhibitory molecule expression (F). The percentage of PD-1⁺ cells (G) and PD-1 level (mean fluorescence intensity [MFI]) on the surface of individual CD4⁺ polyfunctional T cells (H) isolated from the spleen and the liver of Balb/c recipients 6 and 8 days after HSCT. (I-J) Plots and graphs show the percentage of PD-L1–expressing donor CD4⁺ T cells isolated from the spleen and liver of Balb/c recipients. ∗P < .05; ∗∗∗P < .001, Student t test. At least 3 mice per group were analyzed.

Figure 4.

PD-1 blockade restored the GVHD-inducing capacity of Id3-cKO T cells. (A-E) Balb/b mice were subjected to total body irradiation (5 Gy on day −1 and 5 Gy on day 0) followed by infusion of 5 × 10⁶ B6 × B6/SJL F1(CD45.1⁺CD45.2⁺) TCD-BM alone or together with 1 × 10⁶ B6 (CD45.1⁻CD45.2⁺) naïve CD4⁺ and 5 × 10⁵ B6 (CD45.1⁻CD45.2⁺) naïve CD8⁺ WT or Id3-cKO T cells. Anti–PD-1 antibody (200 μg per mouse per injection) was administered intraperitoneally on days 0, 3, 5, and 7 after HSCT. In some experiment, isotype immunoglobulin G (IgG) was injected as control. (A) Survival rates of Balb/b mice. (B) Seventeen days after HSCT, cells were isolated from the spleen, liver, and intestine of recipients, enumerated and stained for flow cytometry analysis. (C) Graphs show the percentage of PD-1⁺ donor CD4⁺ T cells in the spleen and liver. (D) PD-1 protein levels on the surface of donor CD4⁺ T cells isolated from the spleen and liver of Balb/b recipients. (E) The number of IFN-γ⁺GM-CSF⁺ population in the spleen and liver. (F) Balb/c transplantation was performed as described in Figure 1. Anti-PD1 antibody was administered with the same regimen as in Balb/b recipients. Graph shows the survival rate. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001, Student t test.

Figure 5.

Characterization of liver infiltrated donor CD4 T cells using scRNA-seq. Allo-HSCT was performed as described in Figure 1. Three weeks after HSCT, livers were harvested from recipients for T-cell enrichment. (A) Uniform manifold approximation and projection for dimension reduction (UMAP) analysis of single-cell transcriptome from liver infiltrated WT and Id3-cKO donor CD4⁺ T cells identified 6 clusters: 1A, 1B, 3A, 3B, 6, and 7. (B) Expression of effector molecules in WT and Id3-cKO CD4⁺ T cells in clusters 1A, 3A, and 3B. The sizes of balls represent percentage of cells expressing the molecule in that cluster, whereas the shades of red represent the average level of the molecule in the cluster. (C) Expression of inhibitory molecules in WT and Id3-cKO CD4⁺ T cells in clusters 1A, 3A, and 3B. (D) Characterization of WT cluster 1A using ingenuity pathway analysis of DEGs in 1A compared with the average transcriptome of other clusters. (E) Ingenuity pathway analysis characterization of Id3-cKO cluster 1A relative to WT cluster 1A.

Figure 6.

Id3 restricts chromatin accessibility at specific gene loci. Naïve CD4⁺ T cells were cultured under Th1 polarization condition for 4 days. CD62^lo effector cell population were sorted for ATAC-seq and RNA-seq libraries preparation. (A) differential ChrAcc sites were assigned to genes less than 50 kb upstream and downstream. DEGs associated with opened chromatins in Id3-cKO Th1 cells were subject to gene ontology (GO) term analysis. (B) Motif analysis of opened chromatin sites in Id3-cKO Th1 cells. (C) Differential accessible windows along the gene Pdcd1 regulatory loci. Data were collected from 3 independent experiments. ATF, activating transciption factors; IRF, interferon regulatory factor; mRNA, messenger RNA.

Figure 7.

ID3 ablation in human T cells reduces xeno-GVHD progression while preserving antitumor activity. ID3 was knocked out from human T cells (including both CD4⁺ T and CD8⁺ T cells) using CRISPR-Cas9 technology and maintained in culture for 7 to 9 days. NSG mice were IV infused with T-cell depleted peripheral blood mononuclear cells (TCD-PBMCs; 10 × 10⁶ cells per mouse) alone or together with WT or ID3-CRISPR-KO human CD4⁺ T cells (10 × 10⁶ cells per mouse) and CD8⁺ T cells (10 × 10⁶ cells per mouse). (A) Survival rates of NSG mice receiving WT and ID3-CRISPR-KO human T cells. (B) The overall survival of leukemia-bearing mice treated with or without WT or ID3-CRISPR-KO CD19-CAR T cells (n = 8-12). (C-F) Balb/c mice were subjected to total body irradiation (4.5 Gy on day −1 and 4 Gy on day 0) followed by infusion of 5 × 10⁶ B6 TCD-BM alone or together with 3 × 10⁵ B6 (CD45.1^-CD45.2⁺) naïve WT or Id3-cKO CD4⁺ T cells and 4 × 10⁵ WT or Id3-cKO CD8⁺ T cells. Recipient mice were challenged with luciferase-expressing P815 mastocytoma cells or A20 lymphoma cells before T-cell infusion. (C) Survival probability. (D) Representative images of tumor luciferase activity detected with live animal in vivo imaging system (IVIS) on days 7, 14, and 34 after transplantation. (E) Summary of GVHD death and tumor death in allo-HSCT mice challenged by P815 cells and A20 cells. (C-E) All Balb/c mice receiving TCD-BM died from tumor; WT donor T cells induced antitumor activity with prolonged median survival time (27.5 days), but all succumbed to tumor and/or GVHD by day 38; in contrast, donor T cells lacking Id3 preserved beneficial GVT effects, leading to significantly improved survival of tumor mice undergoing allo-HSCT. (F) Survival of animals challenged with A20 lymphoma cells. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. GVT, graft-versus-tumor.