Clonal expansion of genome-intact HIV-1 in functionally polarized Th1 CD4+ T cells

Abstract

HIV-1 causes a chronic, incurable disease due to its persistence in CD4+ T cells that contain replication-competent provirus, but exhibit little or no active viral gene expression and effectively resist combination antiretroviral therapy (cART). These latently infected T cells represent an extremely small proportion of all circulating CD4+ T cells but possess a remarkable long-term stability and typically persist throughout life, for reasons that are not fully understood. Here we performed massive single-genome, near-full-length next-generation sequencing of HIV-1 DNA derived from unfractionated peripheral blood mononuclear cells, ex vivo-isolated CD4+ T cells, and subsets of functionally polarized memory CD4+ T cells. This approach identified multiple sets of independent, near-full-length proviral sequences from cART-treated individuals that were completely identical, consistent with clonal expansion of CD4+ T cells harboring intact HIV-1. Intact, near-full-genome HIV-1 DNA sequences that were derived from such clonally expanded CD4+ T cells constituted 62% of all analyzed genome-intact sequences in memory CD4 T cells, were preferentially observed in Th1-polarized cells, were longitudinally detected over a duration of up to 5 years, and were fully replication- and infection-competent. Together, these data suggest that clonal proliferation of Th1-polarized CD4+ T cells encoding for intact HIV-1 represents a driving force for stabilizing the pool of latently infected CD4+ T cells.

Figure 1. HIV-1 DNA levels in highly purified populations of functionally polarized memory CD4⁺ T cells.

(A) Relative proportions of memory CD4⁺ T cells with the indicated functional polarization in HIV-1–infected individuals with acute infection, chronic untreated progressive infection, and cART-treated infection; in HIV-1 controllers; and in uninfected individuals (HIV neg). Significance was tested using a Kruskal-Wallis test with Dunn’s multiple comparisons test. (B) SPICE diagrams reflecting proportions of polarized memory CD4⁺ T cells with indicated cytokine secretion profiles in the different study cohorts. (C) Per-cell levels of HIV-1 gag DNA in sorted memory CD4⁺ T cells from cART-treated individuals with the indicated functional polarization. Empty symbols reflect data points at calculated limits of detection. Significance was tested using a Friedman test with Dunn’s multiple comparisons test. (A and C) *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2. Near-full-length single-template amplification of HIV-1 DNA in CD4⁺ Th cell populations with distinct functional polarization.

(A) Diagrams reflecting the spectrum of HIV-1 DNA products amplified from memory CD4⁺ Th cell populations with indicated functional polarizations, and from unstimulated PBMCs and in ex vivo isolated unfractionated CD4⁺ T cells. Y axis reflects analyzed cell samples and years of cell sampling. Numbers in parentheses indicate absolute frequency of analyzed sequences in each cell population. PCR products with major deletions that were not sequenced were omitted from these diagrams. (B) Pie charts reflecting the relative proportions of the indicated HIV-1 amplification products in cell populations analyzed cross-sectionally. Pooled data from all 3 analyzed patients are shown. (C) Pie charts reflecting contribution of differentially polarized CD4⁺ T cell populations to total number of indicated HIV-1 sequences. Significance was calculated using a Fisher’s exact test; nominal P values are indicated. In B and C, the numbers above the individual pie charts reflect total number of sequences included in each diagram. Hypermut, hypermutations.

Figure 3. Clusters of identical intact HIV-1 proviruses in CD4⁺ T cell populations from cART-treated patients.

(A) Pie charts reflecting relative proportions of defective or intact proviral sequences detected once or multiple times in the indicated cell populations. Pooled data from all 3 study subjects are shown. (B) Pie charts reflecting relative contribution of indicated polarized CD4⁺ T cell populations to total number of clonal and non-clonal sequences within intact and defective proviruses. Significance was calculated using a Fisher’s exact test; nominal P values are indicated. In A and B, numbers above the individual pie charts reflect total number of sequences included in each diagram.

Figure 4. Phylogenetic analysis of near-full-length HIV-1 sequences derived from individuals in chronic and acute HIV-1 infection.

(A) Vertical phylogenetic trees of intact, near-full-length proviral sequences from individual study subjects with chronic HIV-1 infection. Sequences retrieved from PBMCs collected at longitudinal time points are marked with the year of sampling. Sequences without an indicated year of sampling were collected in 2016. Shaded areas indicate clusters of intact proviruses that are completely identical. Viral sequences retrieved from MOLT-4 cells cocultured in Transwells of viral outgrowth assays (VOA) are indicated for subject 1. (B) Vertical phylogenetic tree of intact, near-full-length HIV-1 sequences derived from PBMCs from 2 individuals identified in acute HIV-1 infection.