PMC7596914_01
HE staining of lymph nodes. A) Pulmonary hilar lymph node. Note
expansion of extrafollicular areas. Lymph follicles are not abundant and only focally
observed (inset). B) Retroperitoneal lymph node as a control.
C) Mid-power and D) high-power views of the
extrafollicular area of the pulmonary hilar lymph node, showing proliferation of round
cells (plasmablasts). Inset in D) shows small lymphocytes observed in
the paracortex of pulmonary hilar lymph nodes as size controls at the same
magnification. Scales: 500 mum (A, B), 50 mum
(C), and 10 mum (D including the inset).
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Microscopically, enlarged lymph nodes sampled from the pulmonary hilar area had the following features (Fig. 1A): 1) expansion of interfollicular areas, 2) no increase in germinal centers, and 3) vascular transformation of lymphatic sinuses. Retroperitoneal or mesenteric lymph nodes demonstrated no notable microscopic changes (Fig. 1B). Higher magnification in pulmonary hilar nodes revealed that interfollicular areas were filled with diffuse proliferation of round cells with scant amphophilic cytoplasm, which were larger (up to two-times) than small lymphocytes (Fig. 1C, D, Fig.
Immunohistochemistry for round cells (plasmablasts) in the extrafollicular areas of the
pulmonary hilar nodes, labeled by antibodies against A) CD19,
B) CD20, C) IgG, D) IgM,
E) kappa chain, and F) lambda chain. Plasmablasts are
CD19+ CD20- IgG+ IgM-. Sparse
CD20+ cells were judged to be reactive B cells. No monotypia was observed
for kappa and lambda chains. Scales: 20 mum (A-D).
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However, the immunohistochemical results were unexpected: most round cells in the interfollicular areas were CD3- CD5- CD19+ CD20- CD79a- Pax-5- CD138+ (up to 50%), MUM1+, LMP1-, EBNA2-, and cytoplasmic IgG+ IgA-, and IgM- (Fig. 2A-D, Fig. Kappa and lambda light chain staining did not demonstrate monotypia (Fig. 2E, F).
Multi-labeling immunohistochemistry for phenotypic characterization of plasmablasts in
the pulmonary hilar lymph nodes. A) Chromogenic method.
Ki-67+ cells (brown) did not correspond to CD20+ (red) or
CD3+ (green) cells. B), C)
Immunofluorescence method. Merged figures showing that IgG+ cells
(B, green) and VS38c+ cells (C, green)
were frequently double-positive for Ki-67 (red). Note that Ki-67 is positive in the
nucleus (located in the center). Round cells in the extrafollicular areas were finally
identified as plasmablasts. Scales: 20 mum (A-C).
Methods are described in the legends of Supplementary Figure 5.
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First, we confirmed that neither CD3+ T nor CD20+ B cells in the expanded interfollicular area correspond to Ki-67+ cells (Fig. 3A, Fig. In sharp contrast, immunofluorescence staining revealed that both cytoplasmic IgG+ cells and VS38c+ cells frequently co-expressed Ki-67 (78% among IgG+ cells) (Fig. 3B, C).