Abstract
T follicular helper (Tfh) cells are increasingly recognized as a major reservoir of HIV infection that will likely need to be addressed in approaches to curing HIV. However, Tfh express minimal CCR5, the major coreceptor for HIV-1, and the mechanism by which they are infected is unclear. We have previously shown that macaque Tfh lack CCR5, but are infected in vivo with CCR5-using SIV at levels comparable to other memory CD4+ T cells. Similarly, human splenic Tfh cells are highly infected with HIV-1 DNA. Therefore, we set out to examine the mechanism of infection of Tfh cells. Tfh and other CD4+ T cell subsets from macaque lymph nodes and spleens, splenic Tfh from HIV+ subjects, and tonsillar Tfh from HIV-uninfected subjects were isolated by cell sorting prior to cell surface and molecular characterization. HIV proviral gp120 sequences were submitted to genotypic and phenotypic tropism assays. Entry of CCR5- and CXCR4-using viruses into Tfh from uninfected tonsillar tissue was measured using a fusion assay. Phylogenetic analysis, genotypic, and phenotypic analysis showed that splenic Tfh cells from chronic HIV+ subjects were predominantly infected with CCR5-using viruses. In macaques, purified CCR5+PD-1intermediate(int)+ memory CD4+ T cells were shown to include pre-Tfh cells capable of differentiating in vitro to Tfh by upregulation of PD-1 and Bcl6, confirmed by qRT-PCR and single-cell multiplex PCR. Infected PD-1int cells survive, carry SIV provirus, and differentiate into PD-1hi Tfh after T cell receptor stimulation, suggesting a pathway for SIV infection of Tfh. In addition, a small subset of macaque and human PD-1hi Tfh can express low levels of CCR5, which makes them susceptible to infection. Fusion assays demonstrated CCR5-using HIV-1 entry into CCR5+ Tfh and pre-Tfh cells from human tonsils. The major route of infection of Tfh in macaques and humans appears to be via a CCR5-expressing pre-Tfh population. As the generation of Tfh are important for establishing effective immune responses during primary infections, Tfh are likely to be an early target of HIV-1 following transmission, creating an important component of the reservoir that has the potential to expand over time.
Highlights
T follicular helper (Tfh) cells are a subset of memory CD4+ T cells located in secondary lymphoid tissues such as tonsil, spleen, and lymph node (LN)
We previously obtained cryopreserved single-cell suspensions of splenic tissue from five patients with chronic untreated HIV infection [23], from which CD3+CD4+CD19− cells were sorted into naïve CD45RA+ICOS− and resting memory cell (CD45RA−ICOS−), and two subsets of Tfh defined as (i) CXCR5int+ Tfh (CD45RA−ICOS+PD-1hi+CXCR5int+) and (ii) CXCR5hi+ germinal center (GC) Tfh (CD45RA−ICOS+PD-1hi+CXCR5hi+), as shown in Figure S2 in Supplementary Material
T folliicular helper cells characteristically express the chemokine receptor CXCR5 that directs pre-Tfh, and subsequently Tfh, to B cell areas of organized lymphoid tissue, where they play a critical role in helping GC B cells to produce high-affinity, classswitched antibodies and to develop B cell memory
Summary
T follicular helper (Tfh) cells are a subset of memory CD4+ T cells located in secondary lymphoid tissues such as tonsil, spleen, and lymph node (LN). Tfh cells that have successful interactions with cognate B cells migrate inside the follicle to complete full differentiation and to support the germinal center (GC) reaction [5, 6] This process requires further increases in Bcl expression and several changes in chemokine receptor expression, allowing proper localization into GC as well as high-level expression of adhesion molecules to stabilize cognate T–B interaction. Tfh cells at this stage are sometimes called GC Tfh cells to distinguish them from those located at the T:B border, and those in the follicle but outside GC [5]. We set out to examine the mechanism of infection of Tfh cells
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