Table 1.
Differentiation antigen profiles of B2 B cells, NK cells and NKT cells used in multicolor flow cytometry.
Table 2.
Splenic B2 B cells in mutant C57BL/6 mouse strains.
Table 3.
Impact of T. brucei infection on splenic B2 B cells.
Fig 1.
FACS analysis of splenic B2 B cells in uninfected and T. brucei ANTat 1.1-infected C57BL/6 and Prf1-/- C57BL/6 mice.
(A) Transitional B cells (IgM+AA4.1+B220+) in a representative uninfected C57BL/6 mouse, (B) Transitional B cells in representative day 10 T. brucei infected C57BL/6 mouse; (C)–Marginal zone B cells (IgM+CD21+CD23lo) and follicular B cells (IgM+CD21+CD23hi) in a representative uninfected C57BL/6 mouse, (D) Marginal zone B cells and follicular B cells in a representative day 10 T. brucei infected C57BL/6 mice; (E)—Transitional B cells in a representative uninfected Prf1-/- C57BL/6 mouse, (F) Transitional B cells in a representative day 10 T. brucei infected Prf1-/- C57BL/6 mouse; G Marginal zone B cells and follicular B cells in a representative uninfected Prf1-/- C57BL/6 mouse, (H) Marginal zone B cells and follicular B cells in a representative day 10 T. brucei infected Prf1-/- C57BL/6 mice.
Fig 2.
Splenic B cells and parameters of infection in intact and Prf1-/- C57BL/6 mice infected for up to 30 days with T. brucei AnTat 1.1.
Parasitemia, body weight and blood packed cell volume were monitored in uninfected C57BL/6 and Prf1-/- mice, and mice infected for 10, 20 or 30 days with 5X103 exponentially growing T. brucei AnTat 1.1 (n = 3/group/infection time point). In addition, spleen cells were harvested, stained with mAb to define transitional and mature B2 B cells as described in Table 1 (n = 3/group/post-infection time point, with uninfected controls processed together with infected mice at each time point) and analyzed using flow cytometry using gates shown in S2 Fig. Results are presented as: (A) T. brucei x 106/ml blood (open squares = Prf1-/- C57BL/6, closed circles = C57BL/6), (B) body weight in grams (closed circles = uninfected C57BL/6, open circles = infected C57BL/6, closed triangles = uninfected Prf1-/- mice, open triangles = infected Prf1-/- mice), (C) Blood packed cell volume (PCV) (closed circles = C57BL/6 mice, closed triangles = Prf1-/- mice) measured as the % of blood cell pellet to total volume, (D) total numbers of transitional (types T2+T3) B cells/ spleen, (E) total numbers of Marginal Zone B cells/spleen, (F) total numbers of Follicular B cells/spleen. Data are presented as mean +/- 1 standard deviation. Significance (*<0.05, **<0.001, ***<0.0001) was determined using one-way ANOVA and Tukey’s HSD test comparing uninfected controls to infected individuals within a mouse strain but not between strains. Results are representative of 3 identical experiments.
Fig 3.
Analysis by ELISA and western blotting of trypanosome antigen specific antibodies in sera from intact and Prf1-/- C57BL/6 mice infected for up to 30 days with T. brucei AnTat 1.1.
T. b. brucei AnTat 1.1 lysate (1 μg protein in 0.05% Np40/well) was coated on wells of an ELISA plate and used to detect trypanosome antigen specific antibodies of: (A) IgM, (B) IgG, (C) IgG1, (D) IgG2a classes in dilutions of serum (doubling dilutions from 1:1000 to 1:54,000) from groups of uninfected (naïve) intact C57BL/6 (gray dots) and Prf1-/- C57BL/6 mice (black squares) and mice that had been infected for 10, 20 or 30 days with T. brucei AnTat 1.1(n = 6/group). Trypanosome lysate (0.5% NP40) was subjected to SDS-PAGE, transferred to membrane and stained with pooled serum from a group of uninfected intact or Prf1-/- mice (n = 6), or mice infected with T. brucei AnTat 1.1 for 10, 20 or 30 days as indicated (n = 6/group) to detect polypeptide-specific antibodies of: (E) IgM, (F) IgG1 and (G) IgG2a classes. Results are representative of results obtained in individual mice of each group in 3 identical experiments.
Fig 4.
Distribution of B220+ and MOMA+ cells in spleens of intact and Prf1-/- mice infected with T. brucei AnTat 1.1.
Thin sections (10 μm) of OCT embedded frozen mouse spleens, were air dried, fixed in acetone, rehydrated and stained with anti-B220 (red) to detect B cells and anti-MOMA (green) to detect marginal metallophilic macrophages. The slides were stained and read on the same day on a Zeiss MOT200 inverted microscope with a Zeiss apotome at 20x magnification. Scale bars, 50 micrometers. (A) Uninfected C57BL/6 mice, (B) uninfected Prf1-/- C57BL/6 mice, (C, E) C57BL/6 mice infected for 10 and 30 days with T. brucei AnTat 1.1, (D, F) Prf1-/- C57BL/6 mice infected for 10 and 30 days with T. brucei AnTat 1.1. Results are representative of 5 mice studied in each group.
Fig 5.
Splenic B cell populations in uninfected and T. brucei AnTat 1.1-infected TCR-/- C57BL/6 mice.
Splenic B cells in uninfected B6-TCR-/- mice (n = 3) and B6-TCR-/- (n = 3) mice that had been infected with T. brucei AnTat 1.1 for 10 days were stained for surface markers used to define transitional and mature B2 B cells as described in Table 1 and analyzed using flow cytometry. Results are presented as individual and mean +/- 1SD numbers of: (A) Transitional 2+3 B cells/spleen, (B) MZB cells/spleen and (C) FoB cells/spleen. Significance (*p<0.05; **p<0.01) is determined using one-way ANOVA and Tukey’s HSD test comparing uninfected controls to infected individuals. Results are representative of 3 identical experiments. D–G Representative FACS plots of: (D) FoB and MZB spleen cells from an uninfected TCR-/- C57BL/6 mouse, (E) FoB and MZB spleen cells from a day 10 infected TCR-/- C57BL/6 mouse, (F) Transitional (AA4.1+, B220+) spleen cells from an uninfected TCR-/- C57BL/6, (G) Transitional (AA4.1+, B220+) spleen cells from an a day 10 infected TCR-/- C57BL/6 mouse.
Fig 6.
Efficacy of NK1.1 cell depletion and its impact on splenic B2 B cell survival in C57BL/6 mice infected with T. brucei AnTat 1.1.
(A) NK1.1+ CD3+ (NKT) and NK1.1+ CD3- (NK) cells in spleens of C57BL/6 mice (n = 3) administered 500ug mAb PK136 anti-NK1.1 monoclonal antibody (upper panel) or C57BL/6 mice (n = 3) administered an irrelevant IgG2a monoclonal antibody (lower panel; MP144 Sigma) ip on days 0, 3, and 7 and collected on day 10, (B-D) mean +/- 1 SD and individual total numbers of: (B) splenic T1 and T2 transitional type B cells, (C) marginal zone B cells and (D) follicular B cells in uninfected or T. brucei AnTat 1.1 infected C57BL/6 mice treated with anti-NK1.1 or control mAb for 10 days. Significance (*<0.05) was determined using one-way ANOVA and Tukey’s HSD test comparing uninfected controls to infected individuals. Results are representative of 4 identical experiments.
Fig 7.
T. brucei AnTat1.1 infection-induced changes in NK cell (CD3- NK1.1+) differentiation antigen expression.
Blue lines present results obtained with NK cells from an uninfected mouse and red lines present results obtained with cells from a day 10 T. brucei AnTat 1.1 infected mouse. Results are representative of 3 mice in each experimental group and of 3 identical repeat experiments. (A) CD107a splenic NK cells [CD3- NK1.1+], (B) CD107a splenic NKT cells [CD3+ NK1.1+], (C) CD107a splenic T cells [NK1.1- CD3+] (D) NKp46 spleen, (E) CD49b spleen, (F) Ly49 pan (IFCH) spleen, (G) Ly49H spleen, (H) CD122 spleen, (I) CD11c spleen, (J) TRAIL spleen, (K) FasL spleen.
Fig 8.
Depletion of splenic B cells in T. brucei AnTat 1.1 infected Prf1-/- mice by adoptively transferred C57BL/6 splenic NK cells.
Prf1-/- mice were injected ip with 5x103 T. brucei Antat 1.1 (n = 6; infected) or with PBS (n = 3; uninfected). Five days later, 3 of the infected mice were injected iv., with 5x106 purified C57BL/6 splenic NK cells in 0.2ml PBS/recipient, and the other 3 infected mice were injected iv with 0.2 ml PBS/recipient. Spleens were harvested from recipient mice 96 hours later (corresponding to 9 days after infection), cell suspensions prepared and (A) transitional Type 2 and 3 B cells, (B) follicular B cells, and (C) marginal zone B cells quantified by multicolor flow cytometry. Significance (*<0.05, **<0.01, ***<0.001) was determined using one-way ANOVA and Tukey’s HSD test comparing uninfected controls to infected individuals. Results are representative of 2 identical experiments.
Fig 9.
NK cells selectively kill splenic B cells from mice infected with T. brucei AnTat 1.1.
eFluor 670-labeled B cells from naive mice or mice infected with T. brucei 8 days earlier (105 B cells/well) were incubated at 37°C with or without NK cells from naïve mice or mice infected with T. brucei 9 days earlier to give ratios of between 50 and 0.25 NK cells/1B cell. After 3 hr incubation viable B cells were enumerated in each well. Results are presented as: relative viable B cell recovery, where 1 is the number of viable B cells remaining in cultures after incubation in the absence of NK cells. This was 8 x 104 B cells from uninfected mice/well, and 5 x 104 B cells from infected mice/well). The study was repeated 3 times with similar results.
Fig 10.
NKp46+ and NKp46- NK cells in C57BL/6 mice infected with T. brucei AnTat 1.1 for up to 30 days.
C57BL/6 mice (n = 3/group) were infected by ip inoculation with 5x103 T. brucei AnTat1.1 or sham-infected by ip inoculation of physiologic saline. On days 0, 3, 5, 7, 10, 15, 20, and 30 after infection groups of infected and uninfected mice were killed, spleen cell suspensions made and analyzed by multicolor flow cytometry to identify NKp46+ and NKp46- NK cells (CD3-NK1.1+) using FACs gates shown in S6 Fig. Data are presented as mean +/- 1 standard deviation; dark bars NKp46+ light bars NKp46-.
Table 4.
Differential expression of selected genes in NK cells purified by FACS from pooled spleens (n = 5) of mice infected 10 days earlier with 5 x 103 T. brucei ANTat 1.1E, or sham infected by administration of PBS.