Type I IFN response associated with mTOR activation in the TAFRO subtype of idiopathic multicentric Castleman disease

Abstract

The TAFRO clinical subtype of idiopathic multicentric Castleman disease (iMCD-TAFRO) is a rare hematologic illness involving episodic disease flares of thrombocytopenia, anasarca, fever, reticulin myelofibrosis, renal dysfunction, and organomegaly (TAFRO) and progressive multiple organ dysfunction. We previously showed that the mTOR signaling pathway is elevated in lymph nodes of iMCD-TAFRO patients and that an mTOR inhibitor is effective in a small cohort of patients. However, the upstream mechanisms, cell types, and mediators involved in disease pathogenesis remain unknown. Here, we developed a targeted approach to identify candidate cellular drivers and mechanisms in iMCD-TAFRO through cellular and transcriptomic studies. Using paired iMCD-TAFRO PBMC samples collected during flare and remission, we identified T cell activation and alterations in NK cell and monocyte subset frequencies during iMCD-TAFRO flare. These changes were associated with increased Type I IFN (IFN-I) response gene signatures across CD8+ T cells, NK cells, and monocytes. Finally, we found that IFN-β stimulation of monocytes and T cells from iMCD-TAFRO patient remission samples induced increased mTOR activation compared with healthy donors, and this was abrogated with either mTORC1 or JAK1/2 inhibition. The data presented here support a potentially novel role for IFN-I signaling as a driver of increased mTOR signaling in iMCD-TAFRO.

Keywords: Cellular immune response; Cytokines; Hematology; Immunology; T cells.

Conflict of interest statement

Conflict of interest: DCF receives research support from EUSA Pharma for the ACCELERATE study (formerly sponsored by Janssen Pharmaceuticals) and study drug from Pfizer for NCT03933904 without corresponding financial support. FVR receives research support from Janssen Pharmaceuticals and consultant fees from EUSA Pharma. There is a pending provisional patent application based on the work in this paper.

Figures

Figure 1. Altered number and relative frequency of circulating immune cell subsets during iMCD-TAFRO flare.

(A–C) Clinical blood counts as identified in whole blood at time of remission and flare blood draw (n = 9–10). (A) White blood cell (WBC) count representing PBMCs and neutrophils across paired remission and flare samples (P = 0.0165). (B) Absolute neutrophil count (ANC) in whole blood (P = 0.0069). (C) Absolute lymphocyte count (ALC) in whole blood (P = 0.4785). (D) Absolute monocyte count (AMC) in whole blood (P = 0.0939). (E–F) Relative percent composition of CD4+ T cells, CD8+ T cells, NK cells, B cells, and monocytes across healthy donors (n = 10) (E) and iMCD-TAFRO patients (n = 10) (F), with relative percent composition from flare (on left) and remission (on right) measured by flow cytometry. (G) Cellular composition of major immune cell types by flow cytometry. P values are based on paired 2-tailed t tests between remission and flare samples and unpaired 2-tailed t tests between healthy donor and flare. Proportion of cells were analyzed using compositional analysis (centrometric log-ratio transformation) with Welch t tests for the means (m) and Brown-Forsythe tests for the variance (s2). Data are mean ± SEM. *P < 0.05; **P < 0.01. Bonferroni’s multiple-hypotheses correction was applied to the comparisons for 3 groups, each testing for means and variances (6 independent hypotheses). P values were, therefore, adjusted by multiplication by 6; a correction was not applied for testing across 5 proportions, since these are interdependent and transformed before the statistical test.

Figure 2. Circulating CD8+ T cells are more activated during flare when compared with remission and healthy donors.

(A) Identification of CD8+ T cells (previously gated as singlets, live, CD45+, CD3+) and subsequent gating of nonnaive CD8+ T cells lacking coexpression of CD45RA and CCR7. (B–D) Flow cytometric analyses of healthy donor (n = 10), iMCD-TAFRO flare (n = 9), and iMCD-TAFRO remission (n = 9) nonnaive CD8+ T cells with representative plots gating CD38+HLA-DR+ (B), PD-1+TIGIT+ (C), Ki67+ (D), and perforin+granzymeB+ CD8+ T cells (E) in flare and remission and comparison of frequencies across remission versus flare and healthy donor (HD) versus flare. Data are mean ± SEM. P values are based on paired 2-tailed t tests between remission and flare samples and unpaired 2-tailed t tests between healthy donor and flare with a Bonferroni’s correction for multiple comparisons. *P < 0.05; **P < 0.01.

Figure 3. Circulating CD4+ T cells are more activated during flare when compared with remission and healthy donors.

(A) Identification of CD4+ T cells (previously gated as singlets, live, CD45+, CD3+) and subsequent gating of nonnaive CD4+ T cells lacking coexpression of CD45RA and CCR7. (B–D) Flow cytometric analyses of healthy donor (n = 10), iMCD-TAFRO flare (n = 8), and iMCD-TAFRO remission (n = 8) nonnaive CD4+ T cells with representative plots gating CD38+HLA-DR+ (B), PD-1+TIGIT+ (C), Ki67+ (D), and perforin+granzymeB+ CD4+ T cells (E) in flare and remission and comparison of frequencies across remission versus flare and healthy donor (HD) versus flare. Data are mean ± SEM. P values are based on paired 2-tailed t tests between remission and flare samples and unpaired 2-tailed t tests between healthy donor and flare, with a Bonferroni’s correction for multiple hypotheses testing. *P < 0.05.

Figure 4. Monocyte and NK cell subset composition is altered during flare when compared with healthy donors.

(A) Identification of CD56-expressing and CD16-expressing NK cell subsets, previously gated as singlets, live, CD45+, within the lymphocyte gate (SSC-A-low), CD3–, and CD19– during iMCD-TAFRO flare and remission. (B) Relative frequency of CD56bright NK cells among all NK cells from healthy donors (n = 10), iMCD-TAFRO flare (n = 10), and iMCD-TAFRO remission (n = 10). (C) Identification of monocytes (previously gated as singlets, live, CD45+, within the monocyte gate [SSC-A-intermediate and CD4-intermediate] CD3–, CD19–, CD56–) and subsequent gating of classical (CD14+CD16–) and nonclassical (CD14–CD16+) monocytes during iMCD-TAFRO flare and remission. (D) Ratio of classical to nonclassical monocytes from healthy donors (n = 10), iMCD-TAFRO flare (n = 10), and iMCD-TAFRO remission (n = 10). (E and F) The absolute number of classical (E) and nonclassical (F) monocytes per μL of whole blood across paired remission and flare samples. Data are mean ± SEM. P values are based on paired 2-tailed t tests between remission and flare samples, with a Bonferroni’s correction for multiple comparisons. **P < 0.01.

Figure 5. Circulating immune cell populations have an enriched type I IFN response gene signature during flare when compared with remission.

(A–E) Gene set enrichment plots reporting enrichment of the HALLMARK gene sets with observed FDR q value and normalized enrichment score (NES) across nonnaive CD8+ T cells (A), CD4+ T cells (B), NK cells (C), classical monocytes (D), and nonclassical monocytes (E). Size of points represent number of genes expressed within each HALLMARK gene set. Color representative of P value for each gene set, with P < 0.01 in red, 0.01 < P < 0.05 in green, and P > 0.05 in blue. (F) Heatmap of the top 15 highly expressed genes within the HALLMARK_INTERFERON_ALPHA_RESPONSE reporting the Log2 fold change (LogFC) gene expression between flare and remission across all 3 TAFRO patients and all cell populations in which this pathway was found to be enriched in GSEA. (G) Heatmap reporting the natural LogFC serum levels of HALLMARK_INTERFERON_ALPHA_RESPONSE proteins between flare and remission from TAFRO-1, TAFRO-2, and TAFRO-3 using the SomaLogic platform.

Figure 6. mTORC1 signaling is enriched and associated with type I IFN response gene expression in classical monocytes from iMCD flare.

(A and B) Enrichment of HALLMARK_MTORC1_SIGNALING in classical monocytes (A) and nonclassical monocytes (B). (C and D) Dot plots with linear regression of the average expression of genes from the HALLMARK_MTORC1_SIGNALING gene set and average expression of genes from the HALLMARK_INTERFERON_ALPHA_RESPONSE gene set within each single-cell sequenced from TAFRO-1, TAFRO-2, and TAFRO-3, identified as classical monocytes (C) and nonclassical monocytes (D). Linear regression was used to assess association between 2 continuous variables, and significant association was set if the lower 95% of the CI was above zero.

Figure 7. Induction of mTOR signaling downstream of IFN-β in iMCD-TAFRO.

(A) Schematic detailing Type I IFN proximal signaling to JAK, downstream activation of mTOR to phosphorylate ribosomal protein S6, and parallel engagement of p38 and pSTAT1/pSTAT2 leading to expression of IFN-stimulated genes (ISGs). Created with www.BioRender.com (B–D) Percent of healthy donor (black) cells (n = 8) and iMCD-TAFRO remission (blue) cells (n = 8) expressing phosphorylated ribosomal protein S6 (S235/S236) upon stimulation with 100 ng/mL IFN-β for 0, 30, 60, or 120 minutes within CD14+ classical monocytes (B), CD4+ T cells (C), and CD8+ T cells (D). (E–G) Percent of iMCD-TAFRO remission cells (n = 8) expressing phosphorylated ribosomal protein S6 (S235/S236) upon stimulation with 100 ng/mL IFN-β for 0 or 120 minutes within CD14+ classical monocytes (E), CD4+ T cells (F), and CD8+ T cells (G). (H–J) Comparison of the change in the percentage of cells compared with untreated samples expressing phosphorylated ribosomal protein S6 (S235/S236) following treatment with either 100 ng/mL IFN-β alone or both IFN-β and 1 μM JAKi within CD14+ classical monocytes (H), CD4+ T cells (I), and CD8+ T cells (J) from iMCD-TAFRO samples from remission. Data are mean ± SEM. For time-dependent comparisons, P values are based on 2-way ANOVA with Bonferroni’s correction for multiple comparisons. P values are based on 1-tailed, paired t tests between samples treated with IFN-β and IFN-β and JAKi.