Retroviruses use CD169-mediated trans-infection of permissive lymphocytes to establish infection

Abstract

Dendritic cells can capture and transfer retroviruses in vitro across synaptic cell-cell contacts to uninfected cells, a process called trans-infection. Whether trans-infection contributes to retroviral spread in vivo remains unknown. Here, we visualize how retroviruses disseminate in secondary lymphoid tissues of living mice. We demonstrate that murine leukemia virus (MLV) and human immunodeficiency virus (HIV) are first captured by sinus-lining macrophages. CD169/Siglec-1, an I-type lectin that recognizes gangliosides, captures the virus. MLV-laden macrophages then form long-lived synaptic contacts to trans-infect B-1 cells. Infected B-1 cells subsequently migrate into the lymph node to spread the infection through virological synapses. Robust infection in lymph nodes and spleen requires CD169, suggesting that a combination of fluid-based movement followed by CD169-dependent trans-infection can contribute to viral spread.

Fig. 1. MLV and HIV infection are facilitated by CD169/Siglec1-expressing macrophages

(A) Immunohistochemistry of a pLN section 0.5 hours after s.c. injection of MLV Gag-GFP (green) into a C57BL/6 mouse. Red, B cells; blue, collagen. (B) Image sequence (movie S2) of HIV Gag-GFP (green) capture at pLN SCS floor (collagen, blue) after s.c. injection into C57BL/6 mouse. Time in minutes. (C) Quantification of MLV Gag-GFP capture at pLNs (n=7-9) 1 hour after s.c. injection. MLV-binding (GFP⁺) cell types were identified by the indicated markers. (D) Image of MLV (green) capture by CD169-expressing macrophages (red) at the pLN SCS floor. CD169⁺ macrophages were stained by s.c. injection of CD169-CF568 antibodies. (E and F) Quantification of MLV capture at pLNs 1 hour after s.c. injection into C57BL/6 mice for wild-type (WT, n=10), mutant (ΔEnv, n=10) or ganglioside-depleted MLV Gag-GFP (PDMP⁺, n=5). Total amounts of Gag-GFP were estimated by Western blotting and ganglioside-depletion by cholera toxin B-horseradish peroxidase (CtxB-HRP) staining. (G and H) MLV Gag-GFP (n=4) and HIV Gag-GFP (n=7-8) capture at the pLNs of C57BL/6 mice after injection of CD169 or control (Ctrl) antibodies. (I and J) MLV Gag-GFP (n=6) and HIV Gag-GFP (n=4) capture at the pLNs of C57BL/6 and Siglec1^−/− mice. (K) Immunohistochemistry of a spleen section 0.5 hours after i.v. injection of MLV Gag-GFP (green). Red, CD169; gray, collagen. (L and M) MLV Gag-GFP capture at the spleen after i.v. injection of CD169 antibodies (n=5) or in Siglec1^−/− mice (n=5). (N) HIV Gag-GFP capture at the spleen of humanized BLT mice in absence or presence of CD169 antibodies (n=3-4). (O and P) MLV-infected cells in pLNs (n=6) and spleen (n=5) of C57BL/6 and Siglec1^−/− mice 2 days after infection. (Q) HIV-infected splenocytes in humanized HSC mice injected with CD169 antibodies (n=5). HIV production of ex vivo-cultured splenocytes was quantified using TZM-bl reporter cells. Median is shown in all cases. Kruskal-Wallis test followed by Dunn's posttest (C), (G) and (H); Mann-Whitney test for panels (E), (F) and (I-Q)..

Fig 2. MLV-laden CD169⁺ macrophages form Env-dependent contacts with B-1a cells for trans-infection

(A) Surface marker analysis of MLV-infected B cells in pLNs of C57BL/6 mice 2 days after s.c. infection. (B) Quantification of MLV-infected, adoptively transferred B-1 cells (s.c.) or naïve B cells (s.c., i.v.) (n=4-5). (C) Q-PCR analysis of relative CD169 mRNA expression by pLN-derived CD5⁺ B-1a cells, CD5⁻ B cells and CD169⁺ macrophages. Mean +/− SD shown. (D) Images (movie S5) of MLV (green) capture at pLN SCS floor containing RFP⁺ B-1 cells (red). Asterisk depicts afferent lymphatic vessel entry site. Blue, collagen. Time in minutes. (E) Image sequence (movie S6) of adoptively transferred RFP⁺ B-1 cells (red) and captured MLV Gag-GFP (green) at the pLN SCS floor. Arrows depict cells analyzed in (F). Time in minutes. (F) Instantaneous velocity of representative B-1 cell traces (from Fig. 2E and fig. S15A) in contact with MLV-laden macrophages (red line) or not (black line). (G and H) Track velocities and arrest coefficients of adoptively transferred RFP⁺ B-1 cells in pLNs pre and post s.c. injection of MLV Gag-GFP carrying or lacking Env (+/−Env). Red lines and numbers in (G) are medians. Percentages in (H) are cell population that remained arrested (<2 μm/min) >50% of time. Data from 4 (+Env, 159 tracks), 3 (−Env, 138 tracks) independent experiments. (I) Image sequence (movie S8) of MLV Gag-GFP (green) transfer to RFP⁺ B-1 cell (red) at pLN SCS floor, and B-1 cell instantaneous velocity over time. Asterisk, stable contact (t=0 min); arrows, MLV Gag-GFP transfer to B-1 cell uropod (t=12-15 min). (J and K) Immunohistochemistry and EM overview of a pLN 1 hour after s.c. injection of MLV. (L and M) Electron tomography of a MLV-laden macrophage at the pLN SCS floor and quantification of the cell-virus distance (n=19). Arrowheads depict MLV particles at the surface and within the macrophage. (N - P) Electron tomographies and tomographic 3D reconstruction of synaptic contacts between MLV-laden macrophages and B cells at pLN SCS floor 1 hour after s.c. injection of MLV. Arrowheads depict MLV particles at contact site. Arrows show direct cell-cell contacts between macrophages and B cells (N and O). In the studied tissue sections we observe 2.14 contacts per 1000 μm² (mean, SD=1.2; n=9). Inset in (P) shows continuity between the invagination and a virus-containing compartment. Kruskal-Wallis test followed by Dunn's posttest for panel (B); Wilcoxon matched-pairs signed rank test for panels (G and H; pre vs. post); Mann-Whitney test for panels (G and H; +Env_post vs. −Env_post).

Fig 3. MLV-infected B-1 cells form stable virological synapses in infected pLNs

(A) Image (movie S11) of adoptively transferred RFP⁺ B-1 cells (red) in MLV Gag-GFP (green) infected pLN 2 days post infection. ROIs show Gag polarization (ROI1) and membranous protrusion (ROI2) of MLV-infected B-1 cells. (B and C) Track velocities and arrest coefficients of adoptively transferred RFP⁺ B-1 cells in non-infected and infected pLNs. Lines and numbers in (B) are medians. Percentage in (C) are static cell population that remained arrested (<2 μm/min) >60% of time. Data from 5 independent experiments. Non-infected pLNs, 265 RFP⁺ B-1 cell tracks; MLV-infected pLNs, 48 MLV-infected and 361 non-infected B-1 cells tracks. Median is shown. Kruskal-Wallis test followed by Dunn's posttest. (D) Image sequence (movie S12) and instantaneous velocity of an adoptively transferred RFP⁺ B-1 cell (red, arrowhead) forming stable contact with MLV-infected leukocyte (green, arrow). Asterisk (t=12 min) depicts contact formation. (E) Image sequence (movie S12) of MLV Gag-GFP material release (arrow) from an infected B-1 cell at a virological synapse (asterisk). Time in minutes. (F and G) Electron tomography and 3D reconstruction of pLN 2 days after s.c. infection showing MLV-containing membranous material at contact site between the uropod of MLV-infected donor cells (D) and uninfected target cells (T). Arrowheads indicate viral particles at surface of infected cell. In the studied tissue sections we observe 2.6 membrane protrusion contacts per 1000 μm² (mean, SD=1.1; n=11). 3D reconstruction (G, right panel): green, donor cell uropod; pink, target cell; blue and gold, uropod-associated membranous tubules; red, MLV.