Characteristics of circulating KSHV-infected viroblasts during active KSHV+ multicentric Castleman disease

Abstract

Kaposi sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8-associated multicentric Castleman disease (MCD) is a polyclonal B-cell lymphoproliferative disorder that mainly occurs in immunocompromised hosts. The diagnosis relies on lymph node biopsy demonstrating KSHV-infected cells located in the mantle zone with a marked interfollicular plasma cell infiltration. Infected cells are large cells positive for immunoglobulin M (IgM), λ light chain, and CD38, described initially as infected plasmablasts. We show that IgM+λ+CD38high cells were also detectable in the peripheral blood of 14 out of 18 (78%) patients with active KSHV-MCD and absent in 40 controls. Using immunofluorescence and flow-fluorescence in situ hybridization, we demonstrate that these cells are KSHV infected and express both latent and lytic KSHV transcripts. These KSHV-infected viroblasts (KIVs) harbor a distinct phenotype compared with conventional plasmablasts. We also identified several putative mechanisms of immune escape used by KSHV, because KIVs displayed an overall decrease of costimulatory molecules, with a remarkable lack of CD40 expression and are interleukin-10-producing cells. The identification of this specific and easily accessible KSHV+ circulating population brings new elements to the understanding of KSHV-MCD but also raises new questions that need to be clarified.

Graphical abstract

Figure 1.

Phenotype of circulating IgM⁺λ⁺CD38^high cells during KSHV-MCD flare. (A) P1 PBMCs were isolated using Ficoll gradient and stained using the method described in “Material and Methods.” At least 10⁶ events were acquired. Gating strategy was performed as follows: (1) gating on single cells; (2) exclusion of T cells (CD3⁺) and monocytes (CD14⁺); (3) gating on IgM⁺CD38^high cells; (4) gating on λ⁺ cells; and (5) assessment of CD19, CD20, CD24, CD27, and CD40 expression. Retrogating of the IgM⁺λ⁺CD38^high population was performed on a side scatter/forward scatter (SSC/FSC) dot plot. Positivity threshold was determined on the fluorochrome corresponding isotype (depicted in blue). P1 is shown as a representative example. (B) Percentage of CD19, CD20, CD24, and CD27 positivity among IgM⁺λ⁺CD38^high circulating cells detected in 14 patients with KSHV-MCD.

Figure 2.

Low CD40 surface expression on circulating IgM⁺λ⁺CD38^high cells during KSHV-MCD flare. MFI is normalized on isotype MFI (MFI = CD40 MFI-isotype MFI) and expressed for KIV, conventional plasmablasts, and B and T cells. Median values and IQRs are represented for each plot. ∗∗∗∗P < .0001, Mann-Whitney U test.

Figure 3.

Identification of circulating KIV during KSHV-MCD flare using LNA/vIL-6 flow-FISH. Flow-FISH was performed on the peripheral blood of 6 patients with confirmed KSHV-MCD flare. LNA⁺ and/or vIL-6⁺ cells are represented among CD3⁺ (T cells), CD14⁺ (monocytes), and CD3⁻CD14⁻ cells. Values are given as percentages of positive cells among CD3⁻CD14⁻ cells. LNA⁺ and/or vIL-6⁺ cells are depicted in red in an IgM/CD38 dot plot for each patient, showing that most of the infected cells are IgM⁺ and CD38⁺ cells.

Figure 4.

Surface expression of costimulatory molecules on KIV, conventional plasmablasts, KSHV⁺EBV⁻ BCP1 cell line, and EBV⁺ LCL. Histograms represent the percentage of CD40⁺, CD70⁺, CD86⁺, CD137L⁺, OX40-L⁺, BAFF-R⁺, ICOS-L⁺, and PD-L1⁺ cells among KIV, conventional plasmablasts, KSHV⁺EBV⁻ BCP1 cell line and EBV⁺ LCL. The number of samples tested for each marker is detailed in the manuscript. ∗P < .05, Mann-Whitney U test.

Figure 5.

hIL-6 and hIL-10 intracellular staining on PBMCs from 3 patients with KSHV-MCD flare. PBMCs were left unstimulated and intracellular detection of hIL-6 (top) and hIL-10 (bottom) was assessed in KIV, T cells, B cells, and monocytes. KIV staining is shown for the 3 patients (P1, P3, and P7), whereas staining of T cells, B cells, and monocytes is only shown for 1 representative patient (P1) for the purpose of clarity. hIL-6 and hIL-10 staining was negative in B cells and T cells from the 3 patients tested, whereas P3 and P7 had 15% and 47%, respectively, IL-6⁺ monocytes.