Increased macrophage infection upon subcutaneous inoculation of rhesus macaques with simian immunodeficiency virus-loaded dendritic cells or T cells but not with cell-free virus

Abstract

Information on the establishment of immunodeficiency virus infection through transmission of infected cells is sparse. Dendritic cells (DCs) and T cells may be central to the onset and subsequent spread of infection following mucosal exposure. To directly investigate the consequences of virus being introduced by DCs or T cells, we reinjected ex vivo simian immunodeficiency virus (SIV)-loaded autologous immature DCs and T cells subcutaneously (s.c.) into healthy macaques. s.c. injection of cell-bound virus was used to mirror what may happen if virus-loaded cells pass through an epithelium or perhaps DCs and T cells that immediately entrap cell-free virus, having just crossed an epithelial barrier. Virus load in the plasma was monitored along with combined in situ hybridization and immunohistochemistry to identify the cells replicating virus in the lymphoid tissues. Both DCs and T cells transmitted infection after being pulsed with either wild-type or nef-defective (delta nef) SIVmac239. As seen in animals infected intravenously, replication of delta nef was attenuated compared to that of wild-type virus when introduced in either cell-bound form. Upon examination of the draining lymph nodes (LNs) during the first days of infection, virus-producing CD4(+) T cells predominated in control animals that received s.c. cell-free virus. In dramatic contrast, both SIV-positive macrophages and T cells were detected in the LNs of monkeys infected with cell-associated SIV. Therefore, although both cell-free and cell-associated viruses are infectious, the initial cells amplifying the virus differ. This may have important implications for the subsequent dissemination of infection and/or induction of antiretroviral immunity.

FIG. 1.

Immature DCs exposed to SIVmac239 in vitro transmit infection after s.c. reinjection. (A and B) Immature monocyte-derived DCs from two healthy macaques (F154 and L780) were exposed to SIVmac239 (MOI of 0.1), washed, and cultured overnight in the presence of 10 μg of TT per ml. The next day, the cells were washed again, recounted, and monitored for their ability to transmit infection in vitro (A) and in vivo (B). (A) Virus-loaded DCs were mixed with 174xCEM cells (10⁴ DCs with 10⁵ 174xCEM [CEM] cells [F154 DC-SIV+CEM and L780 DC-SIV+CEM]) or plated alone (F154 DC-SIV and L780 DC-SIV) and cultured for approximately 2 weeks. Starting at day 3 of culture, supernatants were sampled every other day and monitored for the presence of RTase activity. The results are expressed as cpm (10³) of RTase activity per μl of culture supernatant. (B) SIV and TT-bearing DCs (L780, 2 × 10⁶ DC-SIV; F154, 1 × 10⁶ DC-SIV) were s.c. reinjected into the donor animals at three sites proximal to an inguinal LN. Blood samples collected over the ensuing weeks were analyzed for virus RNA levels by bDNA analysis. The numbers of RNA copies per milliliter of plasma are shown. (C) LN and spleen (Sp) cell suspensions isolated from tissues obtained at necropsy of animal F154 were assessed for virus levels by SIV gag PCR. Aliquots of 10⁵ cells were sampled, and the SIV gag DNA was amplified. Standard curve controls representing 0 (lane −), 10¹ (lane 1), 10² (lane 2), 10³ (lane 3), and 10⁴ (lane 4) SIV gag copies were included to estimate SIV gag copy numbers in each test sample.

FIG. 2.

PCR confirmation of infection via DC-associated and cell-free s.c. administered virus. (A) Immature DCs from two healthy macaques (P741 and H917) were exposed to 0.1 MOI of delta nef (P741) or wild-type (H917) SIV and washed, and an aliquot (of the cells injected into the animals [Fig. 3]) was cultured alone (DC) or with 174xCEM cells (DC+CEM) for 2 days (at a DC/174xCEM ratio of 1:10). After culture, the cells were collected and the SIV gag DNA levels evaluated by PCR. The standard curve of controls described for Fig. 1 is included. (B) PBMCs were prepared from blood samples taken 1, 4, and 6 weeks after injection of animals with delta nef virus-infected DCs (K080 and M846), wild-type virus-infected DCs (AR 82 and G544), or cell-free wild-type virus (M806) (Table 2). SIV gag DNA levels were detected by PCR analysis, confirming the presence of infection in all animals compared to the standard curve controls (Fig. 1).

FIG. 2.

PCR confirmation of infection via DC-associated and cell-free s.c. administered virus. (A) Immature DCs from two healthy macaques (P741 and H917) were exposed to 0.1 MOI of delta nef (P741) or wild-type (H917) SIV and washed, and an aliquot (of the cells injected into the animals [Fig. 3]) was cultured alone (DC) or with 174xCEM cells (DC+CEM) for 2 days (at a DC/174xCEM ratio of 1:10). After culture, the cells were collected and the SIV gag DNA levels evaluated by PCR. The standard curve of controls described for Fig. 1 is included. (B) PBMCs were prepared from blood samples taken 1, 4, and 6 weeks after injection of animals with delta nef virus-infected DCs (K080 and M846), wild-type virus-infected DCs (AR 82 and G544), or cell-free wild-type virus (M806) (Table 2). SIV gag DNA levels were detected by PCR analysis, confirming the presence of infection in all animals compared to the standard curve controls (Fig. 1).

FIG. 3.

Transmission of infection by cell-associated versus cell-free virus. (A) Immature monocyte-derived DCs or CD4⁺ T cells were isolated from 17 healthy macaques (as indicated) and exposed to an MOI of 0.1 of SIVmac239 wild-type or SIVmac239 delta nef virus. After an overnight incubation, the cells were collected, washed, and recounted, and 3 × 10⁵ to 1.2 × 10⁶ SIV-bearing DCs or T cells were reinjected into the donor animals proximal to the inguinal or axillary LNs (Table 2). Individual animal numbers are listed in each graph. Four other animals were s.c. injected with the indicated doses of cell-free virus. (B) Six healthy macaques were infected i.v. with either 10² TCID₅₀ of wild-type SIVmac239 or 2.35 × 10⁴ TCID₅₀ of delta nef SIVmac239. Plasma viremia was measured by bDNA analysis for up to 100 days after injection of cell-associated or cell-free virus to reveal the number of SIV RNA copies per milliliter of plasma.

FIG. 4.

Increased numbers of virus-infected macrophages in the LNs of animals receiving cell-associated virus. Combined ISH and IH data for the draining LNs of six representative animals, biopsied 14 days after injection of cell-associated versus cell-free SIV, are shown. Animal I686 received wild-type virus (wt)-infected DCs, J995 received wild-type virus-infected T cells, AR 70 and G402 were injected with cell-free wild-type virus, and AR 80 and AT 88 were i.v. infected with wild-type virus. Frozen tissues were processed for IH against the indicated markers (red), counterstained with hemalaun (purple-blue), and followed by ISH for SIV RNA (black grains). Small CD4⁺ T cells (arrows, I686), large CD4⁺ cells (macrophages or activated T cells; asterisks, I686), and CD169⁺ and CD68⁺ macrophages (arrowheads, I686 and J995) that are positive for SIV are apparent in animals injected with either DC- or T-cell-associated SIV. SIV-positive cells are negative for the DC markers CD208 (I686 and J995) and CD1a (J995). The SIV-positive cells in AR 70 and G402 lacked CD169 but expressed CD4 (asterisk). The CD4⁺ cell expressing SIV in the example shown for AR 70 appears to be intermediate in size and is thus difficult to definitively identify as either a T cell or macrophage. SIV-positive cells in the cell-free SIV-infected i.v. controls (AR 80 and AT 88) are smaller CD4⁺ cells (arrow) with few CD68⁺ cells (arrowhead) that lack CD169. Magnifications, ×40 (CD1a and CD208 panels of J995) and ×100 (all other panels).