BCL6 BTB-specific inhibition via FX1 treatment reduces Tfh cells and reverses lymphoid follicle hyperplasia in Indian rhesus macaque (Macaca mulatta)

Abstract

Background: The BTB domain of B-cell lymphoma 6 (BCL6) protein was identified as a therapeutic target for B-cell lymphoma. This study compared the pharmacokinetics (PK) of the BCL6 BTB inhibitor (FX1) between mice and macaques, as well as evaluating its lymphoid suppressive effect in uninfected macaques with lymphoid hyperplasia.

Materials and methods: Eight uninfected adult Indian rhesus macaques (Macaca mulatta) were used in the study, four animals carrying lymphoid tissue hyperplasia. Plasma FX1 levels were measured by HPLC-MS/MS. Lymph node biopsies were used for H&E and immunohistochemistry staining, as well as mononuclear cell isolation for flow cytometry analysis.

Results: Inhibition of the BCL6 BTB domain with FX1 led to a reduction in the frequency of GC, Tfh CD4⁺ , and Tfh precursor cells, as well as resolving lymphoid hyperplasia, in rhesus macaques.

Conclusions: B-cell lymphoma 6 inhibition may represent a novel strategy to reduce hyperplastic lymphoid B-cell follicles and decrease Tfh cells.

Keywords: B-cell lymphoma 6; T follicular helper; germinal center reaction; lymphoid hyperplasia.

Figure 1.. Pharmacokinetic of BCL6 BTB inhibitor (FX1) in uninfected rhesus macaques and mice.

(A) Schematic diagram of the experimental design. Two Indian rhesus macaques received one dose of FX1 at 25mg/kg S.Q. and then underwent five blood collection at 0, 2,4,6 and 24hr later. 18 CD-1 mice received on FX1 at 25mg/kg I.P. and then underwent six blood collections with each collection including three mice at 0.5, 2,4,6, 8 and 24hr later. Additional 3 CD-1 mice received vehicle were used for collecting blood at 24hr later as the negative control. (B) The plasma FX1 concentrations measured at 0, 0.5, 2, 4, 6, 8 and 24hr after one dose of FX1 injection were presented. (C) The FX1 exposure level in mice and macaques was calculated using the area under the curve (AUC) and presented. Mean and standard error were indicated in panel B and C.

Figure 2.. BCL6 BTB inhibitor (FX1) treatment reduced the frequency of lymphoid Tfh CD4⁺ T cells and its precursor cells.

(A-B) Representative histogram (A) and summary (B) showing the frequency of CXCR5⁺CD4⁺ T cells in the lymph node of healthy control macaques, macaques with lymphoid hyperplasia, and those macaques with lymphoid hyperplasia receiving an 8-day course FX1 treatment (except 7-day course FX1 treatment for one animal receiving FX1 at 25mg/kg). (C) Representative flow plot for the frequency of CXCR5^hiPD1^hi Tfh CD4⁺ T cells and its precursor cells (CXCR5^dimCD4⁺) in the lymph node of four healthy control macaques, macaques with lymphoid hyperplasia, and those macaques with lymphoid hyperplasia receiving an 7- or 8-day course FX1 treatment at 25mg/kg. (D-E) The frequency of Tfh (D) and Tfh CD4⁺ precursor cells (E) in lymph nodes from four macaques with lymphoid hyperplasia at baseline and 48hr after an 7- or 8-day course FX1 treatment, as well as four healthy control macaques. (F) Representative images for the lymph node biopsies from the same adult macaques with lymphoid hyperplasia before (left) and 48hr (right) after FX1 treatment (25mg/kg). Anti-CD3 (green), anti-CD68 (blue), and anti-BCL6 (red) are presented. Images were captured at 200x magnification.

Figure 3.. BCL6 BTB inhibitor (FX1) treatment reduced lymphoid hyperplasia and Ki67⁺ T cells in the germinal center.

Lymph node biopsies were collected from four adult macaques exhibiting lymphoid hyperplasia at baseline and 48hr after an 8-day course FX1 treatment. (A-D) H&E staining of lymph node biopsies at baseline (A&B) and after (C&D) 10mg/kg (C) and 25mg/kg (D) FX1 treatment for an 8-day course are presented. (E) Representative images for H&E stained lymph node tissue sections from healthy rhesus macaques. Images were captured at 100x magnification; Germinal center areas are highlighted in each section (see black ovals). (F-I) Immunofluorescence staining of the lymph node biopsies from macaques with lymphoid hyperplasia before treatment (F&G) and after receiving BCL6 FX1 treatment at 10mg/kg (H) and 25mg/kg (I). (J) Higher mangnification of baseline (pre-treatment) images are shown for the expression of Ki67 in the germinal center T cells. The tissue sections were stained with anti-CD3 (green), anti-CD20 (blue), anti-Ki67 (red) and DAPI (grey). Images were captured at 400x magnification.

Figure 4.. BCL6 BTB inhibition via FX1 treatment did not cause any systemic adverse effect in rhesus macaques with lymphoid hyperplasia.

(A) A schema for FX1 injection, blood collection, and lymph node biopsy sample collection in the study. (B) The frequency of immune response cells in the PBMC and lymph node were analyzed by flow cytometry and presented. (C) The frequency of T cell subsets [CD4⁺, CD8⁺, double positive (DP), double negative (DN)] in the PBMC and lymph node analyzed by flow cytometry were presented. (D) The body weight measured before and after FX1 treatment was presented. (E-F) The white blood cell counts (WBC) (E) and platelet (F) obtained by CBC analysis were presented. Note panel B lists color code for animal data shown in panels B-G.