Distinct Patterns of Bcl-2 Expression Occur in R5- and X4-Tropic HIV-1-Producing Lymphoid Tissue Cells InfectedEx Vivo

Abstract

Most HIV-1 replication occurs in secondary lymphoid tissues in T cells within B cell follicles. Mechanisms underlying the accumulation of HIV-1-producing cells at these sites are not understood. Antiapoptotic proteins such as Bcl-2 could promote follicular CD4(+) T cell survival, contributing to sustained virus production. Tonsils obtained from subjects without known HIV infection were disaggregated and analyzed for Bcl-2 expression in follicular (CXCR5(+)) and extrafollicular (CXCR5(-)) CD3(+)CD4(+) cells by flow cytometry. Additional tonsil cells were cultured with phytohemagglutinin (PHA) and interleukin-2 (IL-2) for 2 days, infected with either CCR5(R5) or CXCR4-tropic (X4) GFP reporter viruses, and analyzed for Bcl-2 expression. In freshly disaggregated CD3(+)CD4(+) tonsil cells, mean florescence intensity (MFI) for Bcl-2 was higher in CXCR5(+) (median, 292) compared to CXCR5(-) cells (median, 194; p=0.001). Following in vitro stimulation with PHA and IL-2, Bcl-2 MFI was higher in both CXCR5(+) cells (median, 757; p=0.03) and CXCR5(-) cells (median, 884; p=0.002) in uninfected cultures compared to freshly isolated tonsil cells. Bcl-2 MFI was higher in GFP(+)CD3(+)CD8(-) R5-producing cells (median, 554) than in X4-producing cells (median, 393; p=0.02). Bcl-2 MFI was higher in R5-producing CXCR5(+) cells (median, 840) compared to all other subsets including R5-producing CXCR5(-) cells (median, 524; p=0.04), X4-producing CXCR5(+) cells (median, 401; p=0.02), and X4-producing CXCR5(-) cells (median, 332; p=0.008). Bcl-2 expression is elevated in R5 HIV-1-producing CXCR5(+) T cells in vitro, which may contribute to propagation of R5 virus in B cell follicles in vivo.

Germinal Center T Follicular Helper Cells Are Highly Permissive to HIV-1 and Alter Their Phenotype during Virus Replication

“…We also previously reported that follicular CD4+ T cells have relatively elevated levels of Bcl-2 expression, suggesting that they could be relatively resistant to killing by either NK cells or CTL (20). Importantly, CD8 depletion studies showed no evidence that lymph node follicular cells from HIV-1-infected individuals were less susceptible to CD8+ cell-mediated killing than extrafollicular cells.…”

“…Cells were gated on the CD3+CD8- population as CD4 is downregulated on HIV-1 producing cells both in vivo (19) and during ex vivo infection with these reporter viruses [(19, 20) Fig. 1A].…”

Tissue reservoirs of HIV

“…Furthermore, several recent studies in SIV/macaques and patients on ART identify a memory subset, the CD4+ T-follicular helper cells (T fh ) in LN follicles bearing CXCR5 and PD-1 as highly enriched for replication competent virus and viral RNA[35,36]. The survival of infected T fh has been attributed to the paucity of virus-specific CTL in LN follicles and to the expression of anti-apoptotic BCL2 by T fh in the setting of chronic HIV/SIV infection [37-39]. Recently, preferential retention of HIV in cells in peripheral blood with T fh markers has also been described[40].…”

T FH in HIV Latency and as Sources of Replication-Competent Virus

“…Increased cell survival may contribute to the persistence of T FH as well. CXCR5+ cells in healthy human tonsils express elevated levels of the anti-apoptotic protein Bcl-2, and in the context of ex vivo R5 infection, Bcl-2 is upregulated in productively infected cells [35]. Interestingly, the depletion of CD8 T cells in untreated SIV infection leads to similar levels of replication in lymphoid memory T FH and memory non-T FH , however the memory T FH contain higher levels of RNA and DNA copies up to 135 days after depletion and after CD8 recovery [18], suggesting possible preferential survival of T FH .…”