RHOA G17V induces T follicular helper cell specification and involves angioimmunoblastic T-cell lymphoma via upregulating the expression of PON2 through an NF-κB-dependent mechanism

Abstract

Angioimmunoblastic T-cell lymphoma (AITL) is a malignant hematologic tumor arising from T follicular helper (Tfh) cells. High-throughput genomic sequencing studies have shown that AITL is characterized by a novel highly recurring somatic mutation in RHOA, encoding p.Gly17Val (RHOA G17V). However, the specific role of RHOA G17V in AITL remains unknown. Here, we demonstrated that expression of Rhoa G17V in CD4⁺ T cells increased cell proliferation and induces Tfh cell specification associated with Pon2 upregulation through an NF-κB-dependent mechanism. Further, loss of Pon2 attenuated oncogenic function induced by genetic lesions in Rhoa. In addition, an abnormality of RHOA G17V mutation and PON2 expression is also detected in patients with AITL. Our findings suggest that PON2 associated with RHOA G17V mutation might control the direction of the molecular agents-based AITL and provide a new therapeutic target in AITL.

Keywords: AITL; NF-κB pathway; PON2; RHOA; mutation.

Figure 1.

Rhoa G17V expression in CD4 + T cells increases cell proliferation. (a) Retroviral Flag-Rhoa WT, Flag-Rhoa G17V expression vectors, or GFP control vectors was transduced into mouse CD4⁺ T cells. Transduction efficiency was indicated as GFP signals by flow cytometry analysis. (b) Flag-Rhoa WT, Flag-Rhoa G17V, and endogenous Rhoa expression in CD4⁺ T cells transduced with retroviral Flag-Rhoa WT, Flag-Rhoa G17V expression vectors, or GFP control vectors were detected by western blot analysis. β-Actin served as a loading control. (c) In vitro CellTrace Violet (CTV) proliferation assay of CD4⁺ T cells transduced with retroviral Flag-Rhoa WT, Flag-Rhoa G17V expression vectors, or GFP control vectors for four days. (d) Annexin V and 7-ADD staining of CD4⁺ T cells transduced with retroviral Flag-Rhoa WT, Flag-Rhoa G17V expression vectors, or GFP control vectors on day three were analyzed by flow cytometric analysis. The P value in (C) and (D) was calculated by ANOVA followed by Dunnett’s test using triplicate samples from two independent experiments. Columns indicate means; bars are the standard error. **P ≤ .01.

Figure 2.

Rhoa G17V induces Pon2 expression through activation of the PI3K/Akt/NF-κB pathway in CD4⁺ T cells. (a) Immunoblots showing expression of Pon2, phosphorylated Akt, phosphorylated IκB kinase, IκKβ, IκBα, and IκBβ in CD4⁺ T cells transduced with retroviral Flag-Rhoa WT, Flag-Rhoa G17V expression vectors, or GFP control vectors. β-Actin served as a loading control. (b) Representative confocal images showing immunostaining of NF-κB p65 (Green) in CD4⁺ T cells transduced with retroviral Flag-Rhoa WT, Flag-Rhoa G17V expression vectors, or GFP control vectors. DAPI (Blue) was used as a nuclear counterstain. Original magnification, ×400. The bar graphs on the bottom are showing the quantification of NF-κB p65 immunostaining in at least 200 counted cells, presented as percentage ± SEM. ** P < .01. (c) Immunoblots showing an increase of NF-κB p65 in the nucleus from Rhoa G17V-expressing CD4⁺ T cells. Whole-cell lysates (WCL) and nuclear lysates from CD4⁺ T cells of the indicated groups were immunoblotted with antibodies as indicated. Histone H3 (His H3) is nuclear, while tubulin are cytosolic. (d) Immunoblots showing Duvelisib suppressed the expression of Pon2, decreased the phosphorylated IκB kinase (IKK) complex, and activated the NF-κB p65 in Rhoa G17V-expressing CD4⁺ T cells. β-Actin served as a loading control. (e) Immunoblots showing BAY and Iκ-B super repressor inhibited NF-κB p65 activation and Pon2 expression in Rhoa G17V-expressing CD4⁺ T cells. β-Actin served as a loading control.

Figure 3.

Knockdown of Pon2 in CD4⁺ T cells suppresses Rhoa G17V-induced cell proliferation. (a) The experimental scheme of knockdown of Pon2 in control or Rhoa G17V-expressing CD4⁺ T cells. GFP was used as a marker to monitor the expression of Rhoa G17V or GFP as control; whereas mCherry signals indicated the expression of Pon2 shRNA-mCherry or mCherry as control. GFP and mCherry double positive cells were gated for further immunophenotypic analysis. (b) Knockdown of Pon2 in control or Rhoa G17V-expressing CD4⁺ T cells was confirmed by western blot analysis. β-Actin served as a loading control. (c) In vitro CellTrace Violet (CTV) proliferation assay of GFP and mCherry double positive CD4⁺ T cells of indicated groups for four days. (d) Annexin V and 7-ADD staining of GFP and mCherry double positive CD4⁺ T cells of the indicated groups on day three were analyzed by flow cytometric analysis. The P value in (c) and (d) was calculated by ANOVA followed by Dunnett’s test using triplicate samples from two independent experiments. Columns indicate means; bars are the standard error. **P ≤ .01.

Figure 4.

Knockdown of Pon2 in CD4⁺ T cells impairs Rhoa G17V-induced Tfh cell polarization and function. (a) Schematic representation of experimental design for adoptive T cell transfer experiments. Knockdown of Pon2 in control or Rhoa G17V-expressing OT-II CD4⁺ T cells were transferred into C57BL/6 mice via retro-orbital injection to characterize the functional consequences. (b) Representative images of the inguinal lymph nodes from the indicated groups one month after adoptive transfer of CD4⁺ T. (c) H&E staining of the inguinal lymph nodes isolated from the indicated groups. The bar graphs on the right are showing the number of follicles of the inguinal lymph nodes in six mice. ** P < .01. Original magnification: ×40. Scale bar: 200 μm. (d and e) Tfh cells (d) and germinal center B cells (e) from C57BL/6 mice after adoptive transfer of the indicated groups were analyzed by flow cytometry. (f) ELISA showing the detection of serum levels of cytokines from C57BL/6 mice after adoptive transfer of the indicated groups. The P values in (C, D, E, and F) were calculated by ANOVA followed by Dunnett’s test using triplicate samples of four mice of each group in two independent experiments. Columns indicate means; bars are the standard error. **P ≤ .01.

Figure 5.

PON2 expression is associated with RHOA G17V mutation in AITLs. (a–c) Immunohistochemical analysis of PON2 distribution in normal human lymph nodes (a) and lymphoma biopsies isolated from patients with AITLs bearing RHOA G17V mutation (b) or without RHOA G17V mutation (c). EliVision Plus two-step immunohistochemical technique with 3–3’ diaminobenzidine (DAB) staining was used. Original magnification: × 400. scale bar: 10 µm. (d) Detection of PON2 in AITL cells by Multiplexed fluorescence immunohistochemistry. Left upper panel shows conventional DAPI staining. Left middle panel shows the single PON2 staining in green. Left lower panel shows the single CD4 staining in red. Right panel shows the multiplexed fluorescence staining of PON2 and CD4. Original magnification: × 200; scale bar: 100 µm. (e) The histogram illustrates the percentage of PON2 immunostaining in AITLs bearing RHOA G17V mutation or without RHOA G17V mutation. AITLs with RHOA G17V mutation showed significantly higher PON2 expression levels than AITLs with RHOA wild-type (WT) (chi-square test, P = .002). (f) The histogram illustrates the percentage of PON2 immunostaining in AITLs bearing IDH2 R172 mutation or without IDH2 R172 mutation. There was no difference in PON2 immunostaining between AITLs with IDH2 R172 mutation or without IDH2 R172 mutation (chi-square test, P = .908).

Figure 6.

PON2 expression correlates with poor prognosis in AITLs. Kaplan–Meier analysis of overall survival for patients with AITL with or without RHOA G17V mutation or PON2 expression.