Early spatial and temporal events of human T-lymphotropic virus type 1 spread following blood-borne transmission in a rabbit model of infection

Abstract

Human T-lymphotropic virus type 1 (HTLV-1) infection causes adult T-cell leukemia/lymphoma (ATL) and is associated with a variety of lymphocyte-mediated disorders. HTLV-1 transmission occurs by transmission of infected cells via breast-feeding by infected mothers, sexual intercourse, and contaminated blood products. The route of exposure and early virus replication events are believed to be key determinants of virus-associated spread, antiviral immune responses, and ultimately disease outcomes. The lack of knowledge of early events of HTLV-1 spread following blood-borne transmission of the virus in vivo hinders a more complete understanding of the immunopathogenesis of HTLV-1 infections. Herein, we have used an established animal model of HTLV-1 infection to study early spatial and temporal events of the viral infection. Twelve-week-old rabbits were injected intravenously with cell-associated HTLV-1 (ACH-transformed R49). Blood and tissues were collected at defined intervals throughout the study to test the early spread of the infection. Antibody and hematologic responses were monitored throughout the infection. HTLV-1 intracellular Tax and soluble p19 matrix were tested from ex vivo cultured lymphocytes. Proviral copy numbers were measured by real-time PCR from blood and tissue mononuclear leukocytes. Our data indicate that intravenous infection with cell-associated HTLV-1 targets lymphocytes located in both primary lymphoid and gut-associated lymphoid compartments. A transient lymphocytosis that correlated with peak virus detection parameters was observed by 1 week postinfection before returning to baseline levels. Our data support emerging evidence that HTLV-1 promotes lymphocyte proliferation preceding early viral spread in lymphoid compartments to establish and maintain persistent infection.

FIG. 1.

IEL isolation. The photomicrograph shows rabbit small intestine, demonstrating layers exposed during separation to obtain the IEL compartment. (A) Duodenum prior to intraepithelial lymphocyte isolation. Magnification, ×100. (B) Duodenum following digestion, with intraepithelial lymphocyte isolation. Magnification, ×100. (C) Duodenum villi predigestion. Magnification, ×400. (D) Duodenum villi postdigestion with exposed IEL. Magnification, ×400.

FIG. 2.

Absolute leukocyte and lymphocyte counts over the course of HTLV-1 infection, based on complete hematological analysis and cell differential counts.

FIG. 3.

Reactive and atypical lymphocyte morphologies. Complete hematological analysis from 500 μl of whole blood was performed by automated cell counting, and cell differential counts were confirmed by counting at least 100 leukocytes from blood smears. Lymphocytes from blood smears were evaluated morphologically based on size and nuclear and cytoplasmic characteristics as normal, reactive (increased overall size and nuclei), and atypical (increased size with cleaved nuclei). Representative lymphocytes from stained blood smears from rabbits with elevated lymphocyte counts at 1 week postinoculation are shown. (A) Normal lymphocyte; (B) reactive lymphocyte; (C and D) atypical lymphocytes with cleaved nuclei. Magnification, ×100 with oil immersion. Bar (panel C), 10 μm.

FIG. 4.

Soluble p19 MA production from PBMC and mononuclear leukocytes from spleen, MLN, and IEL. Blood and tissue-derived mononuclear leukocytes were cultured ex vivo for 24 h in complete medium. Cell-free supernatants were tested by using a commercial ELISA for p19 MA production.

FIG. 5.

Optimization of flow cytometry to test for intracellular Tax. MT2 (Tax⁺) cells were serially diluted in Jurkat T cells (Tax⁻) and tested for intracellular Tax by flow cytometry. Following fixation the samples were analyzed by flow cytometry on a Beckman Coulter EPICS Elite flow cytometer. Control Jurkat cells were less than 1% positive in each trial (the lower limit to the assay).

FIG. 6.

Intracellular Tax in PBMC and mononuclear leukocytes cultured from spleen, MLN, and IEL. HTLV-1 intracellular Tax was measured by flow cytometry in lymphocytes isolated from PBMC and mononuclear leukocytes isolated from the spleen, MLN, and IEL. Control rabbit lymphocytes were less than 1% positive with each trial (the lower limit to the assay).

FIG. 7.

Proviral copy analysis, based on real-time PCR analysis of proviral copy number in PBMC and in tissue-derived mononuclear leukocytes isolated from spleen, MLN, and IEL. HTLV-1 proviral copy number is expressed as the number of infected cells per 1 × 10⁴ PBMC and was calculated as follows: {(HTLV-1 pol copy number)/[(rabbit GAPDH copy number)/2]} × 10,000.

FIG. 8.

Model of early versus later HTLV-1 virus spread and determinants of expression. Following intravenous exposure to HTLV-1-infected cells and prior to a robust acquired immune response, the blood and gut-associated lymphoid microenvironment are permissive for active virus replication (documented as soluble p19 from ex vivo cultured leukocytes and proviral copy numbers from blood-derived leukocytes). Following maturation of the immune responses (2 to 4 weeks postexposure and beyond), cells capable of producing viral antigens (p19 and Tax) in culture are reduced due to immune recognition, resulting in a low circulating proviral copy number. In contrast, the gut-associated tissue microenvironment supports continued virus replication to maintain a tissue reservoir. PVL, proviral load; Tax, intracellular Tax from leukocyte cultures; p19, soluble p19 from leukocyte cultures.