The Majority of HIV Type 1 DNA in Circulating CD4⁺T Lymphocytes Is Present in Non-Gut-Homing Resting Memory CD4⁺T Cells

Abstract

Memory CD4+ T lymphocytes in peripheral blood that express integrins a4ß7 preferentially recirculate through gut-associated lymphoid tissue (GALT), a proposed site of significant HIV-1 replication. Tregs and activated CD4 + T cells in GALT could also be particularly susceptible to infection. We therefore hypothesized that infection of these subsets of memory CD4+ T cells may contribute disproportionately to the HIV-1 reservoir. A cross-sectional study of CD4 + T cell subsets of memory CD45RO + cells in peripheral blood mononuclear cells (PBMCs) was conducted using leukapheresis from eight subjects with untreated chronic HIV-1 infection. Realtime polymerase chain reaction (PCR) was used to quantify total and integrated HIV-1 DNA levels from memory CD4 + T cells sorted into integrin b7 + vs. b7 -, CD25 + CD127 low Treg vs. CD127 high , and activated CD38 + vs. CD38 -. More than 80% of total HIV-1 DNA was found to reside in the integrin b7-negative nongut-homing subset of CD45RO + memory CD4 + T cells. Less than 10% was found in highly purified Tregs or CD38+ activated memory cells. Similarly, integrated HIV-1 DNA copies were found to be more abundant in resting non-gut-homing memory CD4 + T cells (76%) than in their activated counterparts (23%). Our investigations showed that the majority of both total and integrated HIV-1 DNA was found within non-gut-homing resting CD4 + T cells.

HIV DNA Subspecies Persist in both Activated and Resting Memory CD4 + T Cells during Antiretroviral Therapy

“…Somewhat contrary to expectations, but consistent with previous observations from our group and others (13,22), we found total HIV DNA to be present in equivalent proportions across all resting/activated subsets before the commencement of ART (Fig. 2).…”

“…As we had stringently sorted each memory subset to 96% to 99% purity, this result cannot be explained by contamination from the CD38 Ϫ subset. However, the relative contribution of activated memory CD4 ϩ T cells to the cellular HIV reservoir is small, given the much lower number of activated CD4 ϩ T cells compared to their resting counterparts, as we previously found in untreated chronically infected subjects (13).…”

“…Written informed consent was obtained from each participant. Subsets of CD4 ϩ CD45RO ϩ T cells, based on coexpression of the CD38 and HLA-DR activation markers, were purified from cryopreserved patient PBMC by cell sorting using a FACSAria as previously described (13 …”

Computationally efficient multidimensional analysis of complex flow cytometry data using second order polynomial histograms

“…sion of CD381 on activated CD41 or CD81 T cells (8)(9)(10), an important prognostic marker in HIV-1 infection (11). This quantitation was originally made with some difficulty in 2 dimensions (12).…”

“…Similarly, inclusion of CD127 clearly reveals the full population of CD251 CD41 Tregs (13), which are even clearer if first divided using CD45RA/CD45RO. Furthermore, CD41 T cells that normally traffic through gutassociated lymphoid tissue, an important population that is particularly targeted by HIV-1 infection (14,15) express the combination of integrins alpha 4 (CD49d) and beta 7, and are most clearly visualized by first using CD45RA/CD45RO (9,10). However, as the number of parameters increases, the complexity of assigning lymphocytes to clusters of cells of similar phenotype greatly increases.…”