CCL2: a Chemokine Potentially Promoting Early Seeding of the Latent HIV Reservoir

Abstract

HIV infects long-lived CD4 memory T cells, establishing a latent viral reservoir that necessitates lifelong antiretroviral therapy (ART). How this reservoir is formed so quickly after infection remains unclear. We now show the innate inflammatory response to HIV infection results in CCL2 chemokine release, leading to recruitment of cells expressing the CCR2 receptor, including a subset of central memory CD4 T cells. Supporting a role for the CCL2/CCR2 axis in rapid reservoir formation, we find (i) treatment of humanized mice with anti-CCL2 antibodies during early HIV infection decreases reservoir seeding and preserves CCR2/5⁺ cells and (ii) CCR2/5⁺ cells from the blood of HIV-infected individuals on long-term ART contain significantly more integrated provirus than CCR2/5-negative memory or naive cells. Together, these studies support a model where the host's innate inflammatory response to HIV infection, including CCL2 production, leads to the recruitment of CCR2/5⁺ central memory CD4 T cells to zones of virus-associated inflammation, likely contributing to rapid formation of the latent HIV reservoir. IMPORTANCE There are currently over 35 million people living with HIV worldwide, and we still have no vaccine or scalable cure. One of the difficulties with HIV is its ability to rapidly establish a viral reservoir in lymphoid tissues that allows it to elude antivirals and the immune system. Thus, it is important to understand how HIV accomplishes this so we can develop preventive strategies. Our current results show that an early inflammatory response to HIV infection includes production of the chemokine CCL2, which recruits a unique subset of CCR2/5⁺ CD4⁺ T cells that become infected and form a significant reservoir for latent infection. Furthermore, we show that blockade of CCL2 in humanized mice significantly reduces persistent HIV infection. This information is relevant to the development of therapeutics to prevent and/or treat chronic HIV infections.

Keywords: CCL2; CRISPR; CyTOF; human immunodeficiency virus; latency; reservoir.

FIG 1

Tonsillar CD4 T cells produce CCL2 following HIV infection in part mediated through IFI16 and STING signaling. Tonsillar CD4 T cells were purified by negative bead selection and infected with HIV (pNL4-3-GFP) by overlay infection as previously described (19). (A) Production of CCL2 and CCL20 RNA was measured by qPCR and plotted as fold increase relative to uninfected controls following 18 h of overlay infection. (B) CCL2 protein secretion measured by Meso Scale Discovery (MSD) was detected following 24 h after HIV infection in overlay cultures employing tonsillar CD4 cells. Addition of raltegravir (an HIV integrase inhibitor) did not block production of CCL2. (C) Cas9 RNPs and two independent guide RNAs were used to knock out IFI16, STING, and cGAS protein expression in primary tonsillar CD4 T cells (NT: nontargeting guide RNA control). (D) CCL2 protein production was significantly diminished (but not eliminated) in HIV-infected cultures when IFI16 or STING was knocked out. Supernatants were harvested 24 h after infection for MSD analysis of CCL2 production. Fold increase over uninfected control is shown for each knockout culture. Experiments were performed with 4 to 6 independent tonsils. Error bars denote SEM; data were analyzed for significance using ANOVA with Tukey’s multiple comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

FIG 2

CCR2/5⁺ CD4 T cells in the lymphoid tissue exhibit a distinct phenotype compared to their cellular counterparts circulating in blood. Freshly isolated mononuclear cells from tonsil and blood were analyzed by multicolor flow cytometry. (A) Gating strategy for tonsillar memory CD4 T cells. FSC, forward scatter; SSC, side scatter. (B) CCR2⁺ expression is primarily limited to CCR5⁺ memory T cells in tonsil (gated as in panel A). (C) Representative chemokine receptor expression of blood-derived memory cells. (D) CCR2⁺ is expressed on a significant fraction of CCR5⁺ memory CD4 T cells: 28% ± 11% in tonsil (n = 18) and 65% ± 10% in blood (n = 8). Box plots show median, 25% quartiles, and maximum and minimum values. (E) Mass cytometry was used to compare blood-derived and lymphoid tissue-derived CD4 cells across 38 different parameters, and tSNE dimensional reduction visualization was performed (n = 10 for tonsil and blood). Shown are representative tSNE plots, colored by surface CCR2 expression. (F) Selected markers highlight differences between lymphoid tissue- (blue) and blood-derived (red) CCR2/5⁺ cells. The X-axis shows marker signal indicated in the histograms, Y-axis shows normalized counts. (see Fig. S2 in the supplemental material for further comparisons).

FIG 3

Tonsillar CCR2/5⁺ cells express markers of central memory, activation, viral synapse, and tissue homing. Freshly isolated CD4 T cells from tonsil were analyzed for expression of 38 surface markers by mass cytometry. (A) Gating strategy for naive (pregated on CD3⁺, CD4⁺, CD8⁻, CD45RO⁻ live single cells), memory (pregated on CD3⁺, CD4⁺, CD8⁻ live single cells), and CCR2/5⁺ CD4 T cells (pregated on memory as described above); data shown from representative tonsil (n = 10). (B) Comparison of marker expression between populations gated as in panel A (top gray trace, naive; middle blue trace, memory; bottom pink trace, CCR2/5⁺).

FIG 4

Tonsillar CCR2/5⁺ T cells are permissive to X4- and R5-tropic HIV infection. (A) Transwell migration assay of purified tonsillar CD4 cells added to the upper chamber of a 2-μm transwell membrane, with 2 ng/mL CCL2 added to the bottom chamber. After 4 h, the migrating cells (collected from the bottom chamber) were analyzed by flow cytometry and counted. Data are presented as a relative migration (fold increase of chemokine receptor-positive over -negative cell migration normalized to untreated control). (B) Assessment of HIV infection in naive, memory (chemokine receptor-negative), CCR5⁺, or CCR2/5⁺ CD4 T cells after sort purification and infection with 100 ng GFP reporter BaL.NL4-3 HIV-1 (R5-tropic). GFP expression denotes productive infection. (C) Relative percentage of GFP-positive cells as gated in panel B, including NL4-3 HIV-1 (X4-tropic) for comparison (n = 4). (D) R5-tropic envelope causes loss of surface CCR2. CCR5 and CCR2 levels of sorted CCR-negative memory (gray) or CCR2/5⁺ cells (pink) spinoculated with X4- (solid lines) or R5-tropic (dotted lines) HIV and cultured for 24 h. (E) To assess the ability of tonsillar CCR2/5⁺ cells to support latent infection, a previously described primary CD4 T cell latency model was employed (36). Cells sorted as in panel B were spinoculated with NL4-3-luciferase reporter virus and cultured in the presence of ART for 5 days. Cells were then reactivated with anti-CD3/28 beads for 24 h. Virus production after reactivation was measured by quantitating luciferase activity. Data are presented as fold increases in stimulated cells relative to unstimulated cells. Experiments involved the analysis of 4 to 8 independent tonsil preparations. Error bars denote SEM by ANOVA with Tukey’s multiple comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 5

Blockade of CCL2 reduces seeding of the HIV reservoir in humanized mice. (A) BLT mice were humanized as described in the text, and mice with equivalent CD4⁺ T cells at 12 weeks posthumanization were assigned to anti-CCL2 and isotype control groups. (B) Diagram of experimental design illustrating the time course of the experiment. Humanized mice began injections of CCL2-blocking antibody (2H5) or isotype control beginning at 24 h preinfection and every other day thereafter until day 11. Mice were challenged with HIV intrarectally at day 0 and again every 24 h for 5 days. At day 7 the mice were administered ART orally in their chow. ART was maintained for 11 weeks and discontinued at week 12 to begin treatment interruption. Mice were bled weekly following ART interruption and euthanized at week 16 postinfection. (C) Plasma samples were analyzed for HIV RNA by PCR as described in Materials and Methods. Statistical analysis of the contingency table was done by two-sided Fisher’s exact test. (D) Spleen cells were analyzed for both total and integrated HIV DNA levels. Animals that tested positive in either assay were considered positive. Statistical analysis of the contingency table was done by two-sided Fisher’s exact test. (E) The levels of splenic CD4⁺ T cells at 16 wpi were analyzed by flow cytometry and found to be similar in the two groups of mice. (F) Spleen cells were further analyzed for proportions of CCR2/5⁺ in the CD4⁺ subset by flow cytometry. Technical problems occurred in one experiment such that data were not obtained from all the mice. For the remaining mice (n = 16 for the controls and n = 15 for CCL2 blockade), statistical analysis by two-way Student’s t test showed a significant difference between the groups.

FIG 6

Integrated HIV proviral DNA is enriched in CCR2/5⁺ cells from infected individuals. Naive, memory (CCR⁻), CCR2⁺, CCR5⁺, or CCR2/5⁺ CD4 T cells were purified by cell sorting from leukapheresis samples obtained from ART-suppressed, HIV-infected donors. (A) Example of sorting gates from donor cells, pregated on live, single CD3⁺ CD4⁺ cells. (B) Amount of integrated HIV in sorted cell populations, normalized to mitochondrial DNA and expressed as fold increase over the amount detected in naive cells. Cells were sorted as in panel A, genomic DNA was isolated, and provirus was measured using Alu-Gag droplet digital PCR. (C and D) Distribution of HIV provirus among various cell populations from nine ART-suppressed HIV-infected donors. Results are shown as a percentage of total HIV detected from all subsets for each individual (n = 9). Results are grouped by cell type in panel C and separated by individual in panel D. Error bars denote SEM by Friedman ANOVA with Dunn’s test for multiple comparisons. *, P < 0.05; **, P < 0.01.