Trypanosomiasis associated elimination of non-related host antibody responses.

<p>Mice were vaccinated with the commercial DPTa vaccine and boosted after three weeks. 14 days after the vaccine boost, mice were infected with 5000 <i>T. brucei</i> AnTat 1.1E parasite by intra-peritoneal injection, followed 10 days later by an intranasal challenged with 5×10⁶ CFU of <i>B. pertussis/</i>mouse (▪). Control groups consisted of non-vaccinated <i>B. pertussis</i> challenged mice (×), and DPTa vaccinated mice that were challenged with <i>B. pertussis</i> 24 days after the second DTPa boost (□). Mice were sacrificed 3h and 3, 5 and 8, days after challenge and lung homogenates were prepared and plated on the Bordet-Gongou agar plates. CFU's were measured after 72 h incubation. Values are represented as the mean±SD of 3 individual mice per time point.</p

<i>T. brucei</i> induced abrogation of B cell proliferation.

<p>CD19⁺ MACS sorted cells derived form control mice or <i>T.brucei</i> AnTat 1.1E infected mice (day 10 post infection) were incubated for 24 h in the presence of different doses of anti-IgM Fab (A,B), or different doses of LPS (C,D). Proliferation was measure by thymidine incorporation. Results were obtained using spleen cell preparations of four individual mice, and represent the mean % of CPM increase ±SD, with the 100% showing the mean CPM level of non-stimulated cells.</p

Alterations of Marginal Zone (MZ) B cells during <i>T. brucei</i> infections.

<p>MZ B cells were detected using FACS as CD21^HighCD23^Low (R1), IgD^IntIgM^High (R2) or B220⁺CD1d⁺ (R3) on spleen cells derived from non- infected mice (A, upper FACS panel ) or day 10 <i>T. brucei</i> AnTat 1.1E-infected mice (A, lower FACS panel). The decrease in total number of MZ B-cells per spleen was calculated for different time points during infection (B), based on the total amount of cells harvested per spleen at each time point (C). Calculations were performed on cells harvested from 3 individual spleens per time point. Values represent the mean±SD. One of four representative experiments is shown.</p

Infection-induced apoptosis of MZB cells. CD21^HighCD23^Low cells were gated and analyzed for 7AAD and Annexin V staining.

<p>The percentage of positive cells was calculated in MZ B cells from naïve mice, as well as from <i>T. brucei</i> AnTat 1.1E infected mice on days 4, 7 and 10 (A). CD21^HighCD23^Low cells were FACS sorted on day 7, lyzed and analyzed in Western Blot with an anti-caspase 3 antibody, showing bands at 32 kD (pro-caspase 3), and cleaved forms 17 kD and 12 kD (B). RT-PCR for Casp3, BAFF-R, and Bcl-2 was performed on CD19⁺ spleen cells isolated from naïve mice as well as from <i>T. brucei-</i>infected mice on day 4 and day 7 (C). Results present the means of 3 mice per time point ±SD. One of three representative experiments is shown.</p

Destruction of spleen marginal zones.

<p>The MZ macrophage populations of non-infected (upper panel) and day 10 <i>T. brucei</i> AnTat 1.1 infected (lower panel) C57Bl/6 mice are visualized by section staining with ER-TR9 (anti-MZM) (A,C) and MOMA-1 (anti-MMM) antibodies (B,D) (400x magnification).</p

Alteration of Follicular and Plasma B cell numbers during <i>T. brucei</i> infections.

<p>The number of follicular B cells (A) as well as plasma B cells (B) per spleen was calculated on different days after <i>T. brucei</i> AnTat 1.1E infection. Calculations were performed on cells harvested from 3 individual spleens per time point. Values represent the mean±SD. One of four representative experiments is shown. Plasma spleen B cells were stained with a B220/CD138 combination (C).</p

Trypanosomiasis associated elimination of parasite specific host antibody responses.

<p>Survival of mice was recorded after intra-peritoneal infection with of 5000 living parasites, using pleomorphic AnTat 1.1 (A), cloned monomorphic AnTat 1.1 (B), and unrelated cloned monomorphic MITat 1.4 (C) parasites (MS: Median survival). Re-challenge experiments were performed as presented in the insert box. Survival was recorded for the primary pleomorphic AnTat 1.1 infection (▪), and mice re-challenged with the cloned monomrophic AnTat 1.1 (□) or MiTat 1.4 (*) parasites on day 10, in WT mice (D), T-cell deficient nu/nu mice (E), and B-cell deficient µMT mice (F). A re-challenge was subsequently also performed on day 17 in WT mice (G). In each experiment 10 mice per experimental condition were used. One out of three representative experiments is shown for every experimental condition.</p

NK cells selectively kill splenic B cells from mice infected with <i>T</i>. <i>brucei</i> AnTat 1.1.

<p>eFluor 670-labeled B cells from naive mice or mice infected with <i>T</i>. <i>brucei</i> 8 days earlier (10⁵ B cells/well) were incubated at 37°C with or without NK cells from naïve mice or mice infected with <i>T</i>. <i>brucei</i> 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.</p

FACS analysis of splenic B2 B cells in uninfected and <i>T</i>. <i>brucei</i> ANTat 1.1-infected C57BL/6 and <i>Prf1</i>^<i>-/-</i> C57BL/6 mice.

<p><b>(A)</b> Transitional B cells (IgM⁺AA4.1⁺B220⁺) in a representative uninfected C57BL/6 mouse, <b>(B)</b> Transitional B cells in representative day 10 <i>T</i>. <i>brucei</i> infected C57BL/6 mouse; (<b>C)</b>–Marginal zone B cells (IgM⁺CD21⁺CD23^lo) and follicular B cells (IgM+CD21+CD23^hi) in a representative uninfected C57BL/6 mouse, <b>(D)</b> Marginal zone B cells and follicular B cells in a representative day 10 <i>T</i>. <i>brucei</i> infected C57BL/6 mice; <b>(E)</b>—Transitional B cells in a representative uninfected <i>Prf1</i>^<i>-/-</i> C57BL/6 mouse, <b>(F)</b> Transitional B cells in a representative day 10 <i>T</i>. <i>brucei</i> infected <i>Prf1</i>^<i>-/-</i> C57BL/6 mouse; <b>G</b> Marginal zone B cells and follicular B cells in a representative uninfected <i>Prf1</i>^<i>-/-</i> C57BL/6 mouse, (H) Marginal zone B cells and follicular B cells in a representative day 10 <i>T</i>. <i>brucei</i> infected <i>Prf1</i>^<i>-/-</i> C57BL/6 mice.</p

<i>Trypanosoma brucei</i> Co-opts NK Cells to Kill Splenic B2 B Cells

<div><p>After infection with <i>T</i>. <i>brucei</i> AnTat 1.1, C57BL/6 mice lost splenic B2 B cells and lymphoid follicles, developed poor parasite-specific antibody responses, lost weight, became anemic and died with fulminating parasitemia within 35 days. In contrast, infected C57BL/6 mice lacking the cytotoxic granule pore-forming protein perforin (<i>Prf1</i>^<i>-/-</i>) retained splenic B2 B cells and lymphoid follicles, developed high-titer antibody responses against many trypanosome polypeptides, rapidly suppressed parasitemia and did not develop anemia or lose weight for at least 60 days. Several lines of evidence show that <i>T</i>. <i>brucei</i> infection-induced splenic B cell depletion results from natural killer (NK) cell-mediated cytotoxicity: i) B2 B cells were depleted from the spleens of infected intact, T cell deficient (<i>TCR</i>^<i>-/-</i>) and FcγRIIIa deficient (CD16^-/-) C57BL/6 mice excluding a requirement for T cells, NKT cell, or antibody-dependent cell-mediated cytotoxicity; ii) administration of NK1.1 specific IgG2a (mAb PK136) but not irrelevant IgG2a (myeloma M9144) prevented infection-induced B cell depletion consistent with a requirement for NK cells; iii) splenic NK cells but not T cells or NKT cells degranulated in infected C57BL/6 mice co-incident with B cell depletion evidenced by increased surface expression of CD107a; iv) purified NK cells from naïve C57BL/6 mice killed purified splenic B cells from <i>T</i>. <i>brucei</i> infected but not uninfected mice <i>in vitro</i> indicating acquisition of an NK cell activating phenotype by the post-infection B cells; v) adoptively transferred C57BL/6 NK cells prevented infection-induced B cell population growth in infected Prf1^-/- mice consistent with <i>in vivo</i> B cell killing; vi) degranulated NK cells in infected mice had altered gene and differentiation antigen expression and lost cytotoxic activity consistent with functional exhaustion, but increased in number as infection progressed indicating continued generation. We conclude that NK cells in <i>T</i>. <i>brucei</i> infected mice kill B cells, suppress humoral immunity and expedite early mortality.</p></div