Spleen-dependent turnover of CD11b peripheral blood B lymphocytes in bovine leukemia virus-infected sheep

Abstract

Lymphocyte homeostasis is determined by a critical balance between cell proliferation and death, an equilibrium which is deregulated in bovine leukemia virus (BLV)-infected sheep. We have previously shown that an excess of proliferation occurs in lymphoid tissues and that the peripheral blood population is prone to increased cell death. To further understand the mechanisms involved, we evaluated the physiological role of the spleen in this accelerated turnover. To this end, B lymphocytes were labeled in vivo using a fluorescent marker (carboxyfluorescein diacetate succinimidyl ester), and the cell kinetic parameters (proliferation and death rates) of animals before and after splenectomy were compared. We show that the enhanced cell death observed in BLV-infected sheep is abrogated after splenectomy, revealing a key role of the spleen in B-lymphocyte dynamics.

FIG. 1.

A BLV-infected sheep (4535) and a control (4534) were injected intravenously with 25 mg of CFSE before and after splenectomy. Blood was collected by jugular venipuncture before and 15 min, 2 days, and 5 weeks after injection. After PBMC isolation, B lymphocytes were labeled with anti-IgM antibodies, and 20,000 events were collected by use of flow cytometry. The x axis and the y axis correspond to CFSE and B-lymphocyte labeling, respectively. The percentages of CFSE⁻ B⁺ and CFSE⁺ B⁺ cells in the total B-lymphocyte population are indicated in the upper quadrants of the dot plots.

FIG. 2.

CFSE kinetics of B cells in BLV-infected and control sheep. The left and right graphs correspond to the percentages of B cells labeled with CFSE within the B-lymphocyte population measured in short-term (5 days) and long-term (83 days) time intervals, respectively. Nonsplenectomized (A and B) and splenectomized (C and D) sheep were analyzed. CFSE kinetics were performed on sheep infected with wild-type BLV (no. 2091 [▪], 3002 [▬], 4270 [⧫], 4535 [▴], and 4536 [•] solid lines) or with an attenuated G4 mutant (no. 1071 [▬], 1077 [□], 4537 [⧫], and 4538 [+] dashed lines). Uninfected control animals (dotted lines) were 2147 (□), 3004 (×), 4164 (⋄), 4533 (▵), and 4534 (○). Thick arrows label day 27, when the percentages of CFSE-positive B cells of nonsplenectomized BLV-infected sheep reach levels of <5%. Data from unsplenectomized sheep were previously published in reference and are replotted in this work.

FIG. 3.

Differential CFSE kinetics pertains to the CD11b⁺ population. CFSE kinetics were performed on sheep infected with wild-type BLV (no. 2091 [▪], 3002 [▬], 4270 [⧫], 4535 [▴], and 4536 [•] solid lines) or with an attenuated G4 mutant (1071 [▬], 1077 [□], 4537 [⧫], and 4538 [+] dashed lines). Uninfected control animals (dotted lines) were 2147 (□), 3004 (×), 4164 (⋄), 4533 (▵), and 4534 (○). After intravenous CFSE injection in nonsplenectomized (A and B) and splenectomized (C and D) sheep, PBMCs were collected at three selected days and labeled with IgM and CD11b antibodies. Labeled cells were then analyzed by flow cytometry on the basis of 50,000 events. The percentages of cells labeled with CFSE within the B⁺ CD11b⁻ (panels A and C) and B⁺ CD11b⁺ (panels B and D) subpopulations are indicated. Thick arrows label day 27, when the percentages of CFSE-positive B cells of nonsplenectomized BLV-infected sheep reach levels of <5%. Data from unsplenectomized sheep were previously published in reference and are replotted in this work.

FIG. 4.

CFSE kinetics of B-cell subpopulations in splenectomized sheep. CFSE kinetics analyses were performed on splenectomized sheep infected with wild-type BLV (no. 3002 [▬], 4270 [⧫], and 4535 [▴] solid lines) or with an attenuated G4 mutant (1077 [□] and 4538 [+] dashed lines). Uninfected control animals (dotted lines) were 3004 (×), 4164 (⋄), and 4534 (○). After intravenous CFSE injection in sheep, PBMCs were collected at three selected days and labeled with IgM and L-selectin, CD21- or CD5-specific antibodies. Labeled cells were then analyzed by flow cytometry on basis of 50,000 events. The percentages of cells labeled with CFSE within different subpopulations of B lymphocytes are indicated. (A) B⁺ L-selectin⁻; (B) B⁺ L-selectin⁺; (C) B⁺ CD21⁻; (D) B⁺ CD21⁺; (E) B⁺ CD5⁻; (F) B⁺ CD5⁻.

FIG. 5.

Proviral loads (represented as numbers of copies per 100 cells) were measured by real-time PCR and normalized with the amplification of 18S ribosomal DNA. Analyses were performed at days 0, 14, and 55 after CFSE injection. The standard deviations of individual values reflect the variability of the PCR quantification (three repetitions). Proviral loads were quantified in sheep infected with wild-type BLV (no. 2091, 3002, 4270, 4535, and 4536) or with the G4 mutant strain (no. 1071, 1077, 4537, and 4538). For each animal, the three successive values correspond to the proviral loads determined at days 0 (•), 14 (▴), and 55 (▪).

FIG. 6.

Summary of the cell kinetic parameters determined for control sheep (noninfected) and for animals infected with a wild-type BLV provirus or an attenuated strain (G4 mutant) as indicated. Minimal proliferation and death rates were estimated by fitting a theoretical model with two types of flow cytometry data: the percentages of B lymphocytes labeled with CFSE and their relative fluorescence intensities. The proliferation and death rates correspond to the percentages of cells produced by proliferation and those disappearing per day, respectively. Statistical relevance (NS, not statistically significant; **, highly statistically significant) among the death rate parameters was calculated according to the two-tailed unpaired Student t test.