CCR7 ligands CCL19 and CCL21 increase permissiveness of resting memory CD4⁺ T cells to HIV-1 infection: a novel model of HIV-1 latency

The major barrier to eradication of HIV-1 is persistent long-lived and latently infected resting CD4⁺ T cells.^1,2  Preintegration latency refers to unintegrated HIV-1 DNA that is unstable and will either degrade or will integrate into the host cell genome, usually after cell activation.^3,,–6  Postintegration latency refers to the presence of integrated HIV-1 DNA in cells that are not actively producing viral particles. One of the paradoxes of postintegration latency is the inefficiency of reverse transcription and integration of HIV-1 into resting CD4⁺ T cells from the peripheral blood,⁷  whereas there is efficient infection of resting CD4⁺ T cells in lymphoid organ cultures in vitro or in the tissues of HIV-1-infected persons or SIV-infected macaques.^8,,–11 

The 2 known CCR7 ligands, CCL19 and CCL21, are constitutively expressed in lymphoid organs, particularly by resident stromal cells in the T-zone, and are critical for T-cell and dendritic cell (DC) trafficking within secondary lymphoid organs.^12,–14  Given that latent HIV-1 infection predominantly occurs in CCR7 expressing resting CD4⁺ T cells^15,,–18  and that infection of resting CD4⁺ T cells occurs with greater efficiency in lymphoid tissue compared with blood, we hypothesized that CCL19 and CCL21 may be critical factors that condition resting CD4⁺ T cells to HIV-1 infection, integration and latency.

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque (GE Healthcare, Chalfont St. Giles, United Kingdom) density centrifugation. Resting CD4⁺ T cells were obtained by negative selection and magnetic beads (Figure 1A).

Purified resting CD4⁺ T cells (either total or purified CD45RO⁻ (naive) and CD45RO⁺ (memory) T cells) were cultured for 3 days in the presence of CCL19 (10-100 nM) or CCL21 (10-100 nM; R&D Systems, Minneapolis, MN), phytohemagglutinin (PHA; 10 μg/mL)/interleukin-2 (IL-2) (10 U/mL; Boehringer Mannheim, Mannheim, Germany) or left unactivated. In some experiments, an antibody to CCR7, 3D12 (10 μg/mL; BD Biosciences, Franklin Lakes, NJ) or IgG_2a isotype control was added to resting CD4⁺ T cells 24 hours before addition of CCL19. We then infected the cells with either pNL4.3 (X4 using) or AD8 (R5 using) HIV-1 or HIV-1 containing a deletion in the nef gene (Δnef) and replaced with enhanced green fluorescent protein (EGFP) [pNL4.3 Δnef EGFP and AD8 Δnef EGFP] or pNL4.3 with a mutation in integrase D116N¹⁹  (Figure 1B). All infections were performed at a multiplicity of infection of 1 count per minute reverse transcriptase (RT) per cell. RT concentration was determined as described previously.²⁰  In some experiments, integrated HIV-1 DNA was quantified using Alu-long terminal repeat (LTR) real-time polymerase chain reaction (PCR) as described previously.^7,21 

Cells were washed and stained with anti-CD25-phycoerythrin (PE), anti-CD69-fluorescein isothiocyanate (FITC), human leukocyte antigen (HLA)-DR-PE, anti-CCR7-PE, anti-CCR5-PE, and anti-CXCR4-PE (BD Biosciences). Intracellular staining for expression of Ki-67 was performed as described previously.²²  Analysis was performed after 3, 24, 48, and 72 hours in culture using a FACSCalibur flow cytometer (BD Biosciences).

To determine whether integrated HIV-1 DNA was replication-competent, purified resting CD4⁺ T cells were incubated with CCL19 or IL-2/PHA or were left unactivated (as described above) and infected with HIV-1. The cells were then restimulated in the presence or absence of L8 (Merck, White House Station, NJ) using a modified version of a method described previously (Figure 2A).³ 

The Mann-Whitney nonparametric U test was used to determine any significant differences between culture conditions. A P value less than .05 was considered significant.

Highly purified total resting CD4⁺ T cells were incubated with CCL19 or PHA/IL-2 or were left unactivated for 3 days before infection with HIV-1. After infection with pNL4.3, there was low-level production of RT in cells stimulated with CCL19 (n = 6; mean ± SD = 73 ± 26 cpm/mL at day 7 after infection) compared with no RT production in unactivated cells (n = 6; 30 ± 14 cpm/mL; P = .014), and an early peak of productive infection after stimulation with IL2/PHA (n = 6; 1625 ± 461 cpm/mL, P = .025) (Figure 1C). Similar findings were obtained after infection with HIV-1 AD8 (n = 3; Figure 1D). The low-level production of RT was similar whether the cells were incubated with CCL19 or CCL21 (10 nM) alone or in combination (data not shown).

We identified a high concentration of integrated HIV-1 DNA 4 days after infection with pNL4.3 of resting CD4⁺ T cells pretreated with 10 nM CCL19 (n = 5; median [interquartile range, IQR]: 29 000 [3860-78 000] copies per million cells; Figure 1E) compared with infection of unactivated resting CD4⁺ T cells (< 330 copies per million cells; P < .001). It is noteworthy that the frequency of integrated DNA in the CCL19 treated resting CD4⁺ T cells was on average only 6-fold less than in cells pre-treated with PHA/IL2 (n = 5; 200 000 [31 000-250 000] copies per million cells; P = .05). We had similar results after infection with HIV-1 AD8.

We separated resting CD4⁺ T cells into CD45RO⁺- and CD45RO⁻-enriched fractions, and only identified HIV-1 entry and integration in the CD45RO⁺ fraction (n = 2; data not shown). After infection with EGFP-expressing X4 and R5 viruses, we were unable to show any EGFP expression in either the resting or CCL19 conditioned resting CD4⁺ T cells (data not shown), suggesting that the low level RT production was unlikely to be secondary to a small population of productively infected cells. In summary, these data demonstrate high levels of HIV-1 integration and low level RT production in resting CD4⁺ memory T cells after incubation with CCL19 or CCL21

Flow cytometry demonstrated that stimulation with CCL19, CCL21, or both did not alter the expression of any activation markers (HLA-DR, CD25, or CD69), (Figure 1F), and there was no change in intracellular expression of Ki-67 or surface expression of CXCR4 (data not shown). However, in cells incubated with CCL19 or CCL21 compared with unactivated control cells, there was a modest down-regulation of expression of CCR7 (Figure 1F) and a slight increase in CCR5 expression. A decrease in the mean fluorescence intensity (MFI) of CCR7 was identified as early as 3 hours after CCL19 stimulation (data not shown).

Virus replication was induced after stimulation of the HIV-1-infected, CCL19-conditioned resting CD4⁺ T cells (Figure 2A). This was observed in both the presence and absence of an integrase inhibitor (L8) after infection with either AD8 (Figure 2B,C) or pNL4.3 (Figure 2D). We quantified the relative increase in RT 7 days after restimulation compared with cells cultured with the chemokine alone or mitogen, but without a second restimulation. In the CCL19-conditioned cells, after restimulation we observed an increase in RT both in the presence and absence of L8. In contrast, in the unactivated cells, we observed no increase in RT in the presence of L8. There was only an increase in RT in the absence L8 (Figure 2C,D). These findings are consistent with the presence of integrated virus in CCL19 conditioned CD4⁺ T cells but not in unactivated CD4⁺ T cells.

Pretreatment of resting CD4⁺ T cells from blood with CCL19 or CCL21 allows for efficient HIV-1 entry and viral integration and is associated with restricted viral expression consistent with a robust in vitro model for postintegration HIV-1 latency. These studies demonstrate a novel action of the CCR7 ligands to facilitate infection of resting CD4⁺ T cells and establish latency.

This study was supported by National Health and Medical Research Council program grant 358399 (S.R.L.) and American Foundation for AIDS Research grant 106715-40 with support from Concerned Parents for AIDS Research (S.R.L., P.U.C., and S.S.).

Contribution: S.S. carried out the cellular and virologic assays and drafted the manuscript. A.S., F.W., and M.X. participated in coordination of the experiments, developed the real-time assays, and performed initial real-time assays for these experiments. P.U.C. participated in the conception and design of the study, provided supervision for experimental work, provided statistical support, and helped write the manuscript. S.R.L. conceived of the study, participated in the design and coordination, and helped write the manuscript. All authors read and approved of the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Prof Sharon R. Lewin, Infectious Diseases Unit, Alfred Hospital, Level 2, Burnet Building, 85 Commercial Rd, Melbourne, Victoria, 3004, Australia; e-mail:s.lewin@alfred.org.au.