Extended Data Figure 8: Distinct regions of caspase 1 and caspase 3 activity in lymph node of a chronically infected HIV patient.

Inguinal lymph node was collected from a 41-year-old African-American male, infected with an R5-tropic strain of HIV-1. The patient had been on intermittent anti-retroviral therapy between 2004–2009, and stopped anti-retroviral therapy in 2009. This individual exhibited a viral load of 30,173 RNA copies per ml, and CD4 T-cell count of 259 cells per μl. The fresh specimen was immediately subjected to immunostaining in parallel with fresh uninfected human tonsil. Note the immunostain against CD3 highlights the paracortical region, which is almost entirely composed of resting T cells. Note also the sparse presence of CD3-positive T cells in the mantle zones and germinal centres, where lymphocytes become activated (Ki-67) and differentiate into memory and plasma cells. These CD4 T cells are responsible for antigen-dependent activation of B cells in the follicle. Staining for CD11c reveals scattered dendritic cells^67,68 in the germinal centre and largely in the mantle zone. HIV p24 Gag expression is located between the mantle zone and germinal centres, where activated CD4 T cells reside. Remarkably, caspase 3 activity also occurs in this anatomical region, which is separated from the majority of non-productively infected T cells in the paracortical zone and exhibit caspase 1 activation, IL-1β processing and pyroptosis. The anti-caspase 1 antibody was raised against a peptide mapping to the C terminus of caspase 1 p20 of human origin and detects both the cleaved p20 subunit and the precursor of caspase 1. Therefore, in the absence of an equivalent uninfected lymph node it is hard to absolutely determine whether abortive HIV-1 infection affects pro-caspase expression. However, staining of uninfected tonsil or spleen (not shown) tissue revealed no positive HIV p24 Gag, active or pro-caspase 1, bioactive IL-1β or annexin V signals. These data closely correlate with the findings in HIV-infected HLACs where the 95% of the CD4 T cells are non-productively infected CD4 T cells and show activation of intracellular caspase 1, whereas caspase 3 activity is markedly less abundant and specifically occurs in productively infected CD4 T cells. GC, germinal centre; MZ, mantle zone; PC, paracortical zone.