Latent EBV enhances the efficacy of anti-CD3 mAb in Type 1 diabetes

Abstract

Teplizumab is approved for delaying the diagnosis of type 1 diabetes by modulating progression of disease. Compared to EBV-seronegative patients, those who are EBV-seropositive prior to treatment have a more robust response to teplizumab in two clinical trials. Here we compare the phenotypes, transcriptomes and development of peripheral blood cells before and after teplizumab treatment in participants. Higher number of regulatory T cells and partially exhausted CD8⁺ T cells are found in EBV-seropositive individuals than in EBV-seronegative controls at the baseline in the TN10 and AbATE trials. Mechanistically, single cell transcriptomics and functional assays identify the downregulation of NFκB and T cell activation pathways after treatment in EBV-seropositive patients; among diabetes antigen-specific CD8⁺ T cells, T cell receptor and mTOR signaling are also reduced. In parallel, signaling impairment is greater in adaptive than innate immune cells following teplizumab treatment in EBV-seropositive individuals. Our data thus indicate that EBV can impair signaling pathways in immune cells to modulate their responses in the context of type 1 diabetes.

Fig. 1. Changes in immune cell subsets at the baseline among EBV positive or negative individuals.

AViolin plots showing the percentages of populations from the TN10 trial analyzed by flow cytometry and found to be significantly different after FDR correction. Colors identify EBV serological status(n = 27 EBV-seropositive (blue), n = 34 EBV-seronegative (yellow)) (unpaired t tests between EBV positive and EBV negative participants with a two stage linear step up following Benjamin, Krieger and Yekutieli to correct for false discovery rate (FDR) from multiple hypothesis testing, ***p values = 0.0002, 0.00002, 0.0003, 0.0001, 0.00002, 0.00001, 0.0001, 0.00004, 0.0001, 0.0001 in order from left to right in the violin plots, see Supplementary Data 1). B UMAP visualization of the clusters at the baseline. Points represent individual cells and color denote cluster classification as labeled (n = 6 EBV-seropositive (blue) and n = 8 EBV-seronegative (yellow)). C UMAP visualization of the cells at the baseline. Points represent individual cells and color denote EBV serological status (n = 6 EBV-seropositive and n = 8 EBV-seronegative). D Heatmap showing the Z score of the significant pathways (p < 0.05 after FDR correction) in the major clusters at the baseline (n = 6 EBV-seropositive and n = 8 EBV-seronegative). Pathways were inferred based on the DEGs between EBV positive and EBV negative in each cell subset using IPA software. Blue and red scale denote grades of prediction of downregulation or upregulation of the pathways based on the Z score. E Western blot showing phosphorylation of ERK in B cells after stimulation with anti-human IgM. The ratio of pERK/totalERK was corrected for loading (actin). The levels of pERK are decreased in the EBV-seropositive (n = 6) vs EBV-seronegative (n = 5) individuals (mean values ± SEM, Student’s t-test, *p = 0.04). CM: central memory; TEMRA: T effector memory RA; EM: effector memory. Source data are provided in the Source Data file.

Fig. 2. Effects of EBV serostatus on clinical responses to teplizumab.

A Kaplan Meier curve showing the progression from Stage 2 T1D to Stage 3 T1D in TN10 study participants who were EBV-seropositive (n = 18, teplizumab (blue), n = 16 placebo (green)) or EBV-seronegative (n = 26 teplizumab (yellow), 16 placebo (magenta)) at enrollment. Median times to development of Stage 3 T1D: Placebo EBV-seronegative: 12 months; Placebo EBV-seropositive: 35.5 months; Teplizumab EBV-seronegative: 38 months; Teplizumab EBV-seropositive: 86.9 months (Logrank p = 0.005) (See text for subgroup comparisons). B In the AbATE trial (control: n = 16 EBV-seronegative (magenta), n = 7 EBV-seropositive (green), treatment: n = 34 EBV-seronegative (yellow), n = 18 EBV-seropositive (blue)). The data are the least square means of the lnC-peptide(AUC + 1) ( ± 95%CI), corrected for the baseline level and age, from the mixed model. (Control EBV-seronegative vs teplizumab EBV-seronegative (group difference of least square means (LSM, (95% CI): 0.111 (.063, 0.159) p < 0.0001), Control EBV-seropositive vs teplizumab EBV-seropositive (0.19(0.122, 0.258), p < 0.0001). Teplizumab EBV-seronegative vs teplizumab EBV-seropositive (0.066 (0.016, 0.11) **p = 0.008). The annotated comparisons are between teplizumab treated participants who were EBV-seropositive vs EBV-seronegative. Source data are provided in the Source Data file.

Fig. 3. Induction of partially exhausted phenotype CD8⁺ T cells with teplizumab in clinical trial participants.

A Frequency of CD8⁺ Eomesodermin (EOMES)⁺ T cells among CD8⁺ central memory (CM) T cells in the EBV-seropositive (n = 16) vs EBV-seronegative (n = 22) teplizumab treated in the TN10 trial by flow cytometry, (A, **p = 0.004). B Frequency of CD8⁺EOMES⁺ T cells in the EBV-seropositive (n = 10) vs EBV-seronegative (n = 18) teplizumab treated in the AbATE trial by flow cytometry, (**p = 0.004, *p = 0.01). C Frequency of KLRG1⁺ TIGIT⁺ effector memory (EM) CD8⁺ T cells in the TN10 trial among EBV-seropositive (n = 16) vs EBV-seronegative (n = 22) participants by flow cytometry with teplizumab treatment (****p < 0.0001, *p = 0.02). D Frequency of KLRG1⁺ TIGIT⁺ CM CD8⁺ T cells in the EBV-seropositive (n = 16) vs EBV-seronegative (n = 22) participants by flow cytometry with teplizumab treatment (****p < 0.0001, *p = 0.016). E Frequency of KLRG1⁺TIGIT⁺ CD8⁺ T cells in the AbATE trial (*p = 0.02). A–E, the data are the mean values +/− 95% CI from a mixed model for repeated measures without correction for the baseline or multiple comparisons. Control: EBV-seronegative: square magenta, EBV-seropositive: square green; Teplizumab EBV-seronegative triangle yellow, EBV-seropositive inverted triangle blue. The group comparisons are shown in the legends). Source data are provided in the Source Data file.

Fig. 4. Transcriptional changes and pathway analysis of cells from teplizumab treated patients in the TN10 trial who are EBV-seropositive vs EBV-seronegative.

A Heatmap showing the Z score of the pathways with significant differences in the CD8⁺ and CD4⁺ T cells subclusters at the baseline (n = 6 EBV-seropositive and n = 8 EBV-seronegative). The pathways were determined by IPA analysis based on the DEGs between EBV positive and EBV negative in the placebo and teplizumab group. Blue and red scale denote grades of prediction of downregulation or upregulation of the pathways based on the Z score. B Heatmap showing the Z score of the pathways with significant differences (in the CD8⁺, CD4⁺ T cells at 3 months and 18 moths (n = 4 EBV-seropositive, n = 3 EBV-seronegative)). The pathways were determined by IPA analysis and based on the DEGs between EBV positive and EBV negative in the teplizumab group at each timepoint after filtering out the genes that showed changes at the baseline and pathways that change in the placebo group at the same timepoints. Blue and red scale denote grades of prediction of downregulation or upregulation of the pathways based on the Z score. C Volcano plot visualization of the DEGs in EBV positive vs negative related to the mTOR signaling pathway in the CD8 effector cluster at 18 months, and (D) Volcano plot visualization of the DEGs in EBV positive vs negative related to the NFκB in the Treg cluster at 3 months. E Heatmap showing the Z score of the significant pathways in the B cells cluster at 3 months and 18 months. CM: central memory, EM: effector memory.

Fig. 5. Effects of EBV serostatus on autoantigen specific CD8⁺ T cells.

A Bubble plot showing the Z score and p values of the pathways with significant differences in the T1D specific CD8⁺ T cells at the baseline (n = 4 placebo, (EBV-seropositive=2 and EBV-seronegative=2), n = 6 teplizumab, (EBV-seropositive =4, EBV-seronegative =2)) (B) Bubble plot showing the Z score and p values at 18 months in the teplizumab group (n = 3 EBV-seropositive, n = 3 EBV-seronegative). The pathways were determined by IPA analysis based on the DEGs between EBV positive and EBV negative in the T1D specific CD8⁺ T cells of the teplizumab group filtering out the genes that also show a change at the baseline. Blue scale denotes grades of prediction of downregulation of the pathways based on the Z score. C Volcano plot visualization of the DEGs in the T1D specific CD8⁺ T cells between EBV-seropositive vs seronegative at eighteen months in the teplizumab group (n = 3 EBV-seropositive and n = 3 EBV-seronegative).Volcano plot visualization of the DEGs in EBV positive vs negative related to the TCR signaling pathway (D) and TNF signaling (E) in the T1D specific CD8⁺ T cells at 18 months.

Fig. 6. Pseudotime analysis of CD8⁺, CD4⁺ and B cells in the TN10 trial.

UMAPs showing the pseudotime analysis of CD8⁺ T cells in the EBV-seropositive and EBV-seronegative in teplizumab treated participants (A) at all the visits and (B) at 18 months (n = 4 EBV-seropositive n = 3 EBV-seronegative). UMAPs showing the pseudotime analysis CD4⁺ T cells in the EBV-seropositive vs EBV-seronegative participants (C) at all the visits and (D) at 18 months (n = 4 EBV-seropositive n = 3 EBV-seronegative). UMAPs showing the different stages of differentiation of B cells in the EBV-seropositive and EBV-seronegative participants (E) at all the visits and (F) at 18 months (n = 4 EBV-seropositive n = 3 EBV-seronegative). Transcript dynamics are illustrated by the color of the pseudotime. The location of transcriptional signatures for the major cell states identified are indicated by markers on pseudotime visualizations (T _Memory, T_EM, T_Naive, T_Effector, Treg, B _Naive, Plasmablast, B _Memory).