Figure 5. Follicular and extra-follicular T-B conjugates and IgD⁻CD27⁻ B cells in COVID-19 lymph nodes.

(A) Immunofluorescence staining of CD3 (red), CD19 (green) and DAPI (blue) in a lymph node (top) and a spleen (bottom) in late COVID-19 patient. Arrows and arrowheads indicate CD3⁺ T cells and CD19⁺ B cells respectively. T cell and B cells formed close and extensive intercellular plasma membrane contacts. (B) Immunofluorescence staining (left panels) and visualization of T-B conjugates (middle and right panels) in a lymph node from a late COVID-19 patient using the Strata Quest cell-to-cell contact application. Masks of the nuclei based on DAPI staining establish the inner boundary of the cytoplasm and the software “looks” outwards towards the plasma membrane boundary. An overlap of at least 3 pixels of adjacent cell markers was required to establish each “contact” criterion. Details are in the Methods section. Nuclei surrounded by red and green lines respectively depict CD3⁺ T cells and CD19⁺ B cells in T-B conjugates. (C) Representative multi-color immunofluorescence image of CD19 (red), IgD and CD27 (both in green) and DAPI (blue) staining in a lymph node from an early COVID-19 patient. IgD−/CD27− double negative B cells (red staining with no green overlap) are abundant inside the follicle and also outside. Boxed area depicts some of these cells outside the follicle.