Dissemination of bovine leukemia virus-infected cells from a newly infected sheep lymph node

Abstract

To investigate the early establishment of bovine leukemia virus (BLV) infection, we injected BLV-infected or mock-infected allogeneic cells into the shoulder of sheep in which an efferent lymphatic duct of the draining prescapular lymph node had been cannulated. Rare mononuclear cells acting as centers of BLV infection in culture were present within 4 to 6 days in efferent lymph and within 6 to 10 days in blood. Soon after BLV injection, immunoglobulin M+ (IgM+) and CD8+ cells increased in efferent lymph and oscillated reciprocally in frequency. CD8+ blasts increased on days 4 to 6, when infectious centers increased 100-fold in lymph. On days 6 and 7, both lymph and blood were enriched with CD8+ cells that were labeled late on day 5 with an intravenous pulse of 5-bromo-2'-deoxyuridine (BrdU). Lymph, but not blood, was enriched with BrdU+ B cells on day 7. Capsid-specific antibodies became detectable in efferent lymph on days 6 to 8 and surface glycoprotein-specific antibodies on day 9, preceding their detection in serum by 9 to 14 days. Systemic dissemination of BLV-infected cells was thus accompanied by an increase in proliferating CD8+ cells and the onset of BLV-specific antibodies in lymph. Infectious centers reached maximum frequencies of 0.2% in lymph by days 11 to 13, and then their frequencies increased by 5- to 40-fold in blood cells, suggesting that many infected blood cells do not recirculate back into lymph. Beginning on days 10 to 13, a subpopulation of B cells having high levels of surface IgM increased sharply in peripheral blood. Such cells were not present in lymph. After a day 16 pulse of BrdU, recently proliferated cells that stained intensely for surface IgM appeared in blood within 15 h. Predominantly B lymphocytes contained the viral capsid protein when lymph and blood cells were cultured briefly to allow BLV expression. However, both early in lymph and later in blood, BrdU+ B cells greatly exceeded productively infected cells, indicating that new BLV infections stimulate proliferation of two different populations of B cells.

FIG. 1.

Leukocytes in efferent lymph and peripheral blood of cannulated, uninfected sheep 455. Cell counts are plotted in thousands per μl as a function of time after cannulation for efferent lymph cells (A) and leukocytes (B) (closed symbols) and mononuclear cells (open symbols) from peripheral blood. Percentages of live-gated, mononuclear cells staining for the surface markers IgM, CD4, CD8, WC1, and CD14 are plotted for efferent lymph (C) and peripheral blood (D).

FIG. 2.

CA⁺ cells present among efferent lymph cells and PBMCs of BLV-infected animals. Cells immunostained for intracellular CA protein were enumerated by flow cytometry and then plotted as percentages of total live-gated cells. (A) Efferent lymph cells from sheep 454 were stained either directly (fresh) after purification or after being cultured overnight in medium supplemented with LPS. (B) PBMCs from sheep 454 were cultured overnight with fetal bovine serum or with added LPS. (C) Lymph cells from sheep 454 and 972 and (D) PBMCs from sheep 454, 972, and 43 (sham cannulated) were cultured overnight with LPS. Note that different scales are used to plot percentages in individual panels.

FIG. 3.

Infectious centers among cultured, efferent lymph cells and PBMCs from BLV-infected animals. Cells to be tested were suspended in methylcellulose medium supplemented with LPS. Percentages of cells that produced syncytia among indicator cells are plotted as a function of time after sheep were injected with BLV-infected cells. (A and B) Lymph and PBMCs for sheep 454 (A) and 972 (B). (C) PBMCs from sheep 454, 972, and 43 (sham cannulated).

FIG. 4.

Numbers of cells and blasts in efferent lymph and blood. To account for distinctive cell numbers in individual animals, numbers of lymph cells (A) and PBMCs (B) per μl lymph or blood were divided by the average obtained from the same animal during the first 5 days after lymphatic cannulation, before injection of allogeneic cells. The resulting ratios are plotted for BLV-infected sheep 454 and 972 (closed symbols), for mock-infected sheep 973 and 44 (open symbols), and for infected, sham-cannulated sheep 43. In panel A, solid lines depict averages for infected and uninfected animals. (C and D) Blasts were quantified as percentages of live cells by gating on cells with high forward and side scatter. Absolute numbers of blasts per μl are plotted for lymph (C) and blood (D).

FIG. 5.

Cell lineages and blasts among efferent lymph cells of mock-infected and infected sheep. (A) Percentages of live cell labeling on the surface for IgM, CD4, CD8, WC1, and CD14 are plotted for each animal relative to the day of injection with allogeneic cells. Horizontal reference lines represent the average percentage for a given lineage as measured in each animal before injection of allogeneic cells. (B) Reciprocal oscillation of IgM⁺ and CD8⁺ lymph cells in infected, cannulated sheep. (C) Percentages of blasts among lymph cells of different lineages. Blasts were quantified as percentages of lymph cells staining for the indicated surface marker by gating on cells with high forward and side scatter (infected animals, closed symbols; mock-infected animals, open symbols). Averages for infected (solid lines) and mock-infected (dotted lines) animals were calculated using measurements made either on the same day or on adjacent days after injection of allogeneic cells.

FIG. 6.

Cell lineages among PBMCs of mock-infected and infected sheep. Data are presented as described for Fig. 5A with the addition of percentages for sham-cannulated, infected sheep 43.

FIG. 7.

Accumulation of cells with high levels of surface IgM in blood, but not lymph, of BLV-infected animals. (A) Dot plots of surface-stained, live-gated lymph cells (left) and PBMCs (right) are displayed as side scatter (SSC) versus intensity of IgM staining. IgM⁺ cell populations from cannulated sheep 973 are from day 15 after mock infection; cells from infected sheep 454 (day 19) and 972 (day 18) are from the days of peak incidence of infectious centers. (B) Percentages of IgM⁺, IgM^hi, and IgM^mod cells among live-gated PBMCs from BLV-infected and mock-infected sheep were obtained using FloJo software from additional gates drawn around the populations shown in panel A and are plotted as a function of time after injection of allogeneic cells.

FIG. 8.

Percentages of BrdU⁺ cells present among efferent lymph cells and PBMCs after intravenous pulses of BrdU. The percentage of BrdU-stained cells, as determined by flow cytometry, is plotted for the total live-cell population as well as within IgM⁺, CD4⁺, CD8⁺, and WC1⁺ cell subsets. Numbers above each group of bars indicate enrichment (n-fold) for BrdU⁺ cells within a subset relative to the frequency of BrdU⁺ cells within the whole cell population. (A and B) BrdU was injected on day 5 after infection or mock infection, and lymph was collected for staining after 14 h (black bars), 38 h (gray), and 62 h (white). (C) BrdU was injected into sham-cannulated sheep 43 on day 5 after infection and blood was collected 15 h (black bars) and 40 h (gray) later. (D) On day 15, blood was collected from sham-cannulated sheep 43 to evaluate residual BrdU⁺ cells (white bars): relative enrichments within subsets were 0.6 (IgM^hi), 0.8 (IgM^mod), 2.0 (CD4⁺), 1.9 (CD8⁺), and 2.0 (WC1⁺). BrdU was then injected on day 16, and blood was collected 15 h (black bars) and 39 h (gray) later.