T. brucei Infection Reduces B Lymphopoiesis in Bone Marrow and Truncates Compensatory Splenic Lymphopoiesis through Transitional B-Cell Apoptosis

African trypanosomes of the Trypanosoma brucei species are extracellular protozoan parasites that cause the deadly disease African trypanosomiasis in humans and contribute to the animal counterpart, Nagana. Trypanosome clearance from the bloodstream is mediated by antibodies specific for their Variant Surface Glycoprotein (VSG) coat antigens. However, T. brucei infection induces polyclonal B cell activation, B cell clonal exhaustion, sustained depletion of mature splenic Marginal Zone B (MZB) and Follicular B (FoB) cells, and destruction of the B-cell memory compartment. To determine how trypanosome infection compromises the humoral immune defense system we used a C57BL/6 T. brucei AnTat 1.1 mouse model and multicolor flow cytometry to document B cell development and maturation during infection. Our results show a more than 95% reduction in B cell precursor numbers from the CLP, pre-pro-B, pro-B, pre-B and immature B cell stages in the bone marrow. In the spleen, T. brucei induces extramedullary B lymphopoiesis as evidenced by significant increases in HSC-LMPP, CLP, pre-pro-B, pro-B and pre-B cell populations. However, final B cell maturation is abrogated by infection-induced apoptosis of transitional B cells of both the T1 and T2 populations which is not uniquely dependent on TNF-, Fas-, or prostaglandin-dependent death pathways. Results obtained from ex vivo co-cultures of living bloodstream form trypanosomes and splenocytes demonstrate that trypanosome surface coat-dependent contact with T1/2 B cells triggers their deletion. We conclude that infection-induced and possibly parasite-contact dependent deletion of transitional B cells prevents replenishment of mature B cell compartments during infection thus contributing to a loss of the host's capacity to sustain antibody responses against recurring parasitemic waves

The Role of B-cells and IgM Antibodies in Parasitemia, Anemia, and VSG Switching in Trypanosoma brucei–Infected Mice

African trypanosomes are extracellular parasitic protozoa, predominantly transmitted by the bite of the haematophagic tsetse fly. The main mechanism considered to mediate parasitemia control in a mammalian host is the continuous interaction between antibodies and the parasite surface, covered by variant-specific surface glycoproteins. Early experimental studies have shown that B-cell responses can be strongly protective but are limited by their VSG-specificity. We have used B-cell (µMT) and IgM-deficient (IgM−/−) mice to investigate the role of B-cells and IgM antibodies in parasitemia control and the in vivo induction of trypanosomiasis-associated anemia. These infection studies revealed that that the initial setting of peak levels of parasitemia in Trypanosoma brucei–infected µMT and IgM−/− mice occurred independent of the presence of B-cells. However, B-cells helped to periodically reduce circulating parasites levels and were required for long term survival, while IgM antibodies played only a limited role in this process. Infection-associated anemia, hypothesized to be mediated by B-cell responses, was induced during infection in µMT mice as well as in IgM−/− mice, and as such occurred independently from the infection-induced host antibody response. Antigenic variation, the main immune evasion mechanism of African trypanosomes, occurred independently from host antibody responses against the parasite's ever-changing antigenic glycoprotein coat. Collectively, these results demonstrated that in murine experimental T. brucei trypanosomiasis, B-cells were crucial for periodic peak parasitemia clearance, whereas parasite-induced IgM antibodies played only a limited role in the outcome of the infection

Characterizations of B lymphocyte responses during infection with African trypanosomes

Host control of Trypanosoma brucei infections relies on adequate B cell mediated responses including anti-VSG antibody responses. Trypanotolerant animals, namely; Cape buffalo maintain anti-trypanosome specific antibody responses throughout infection. Studies in mice, however; show a failure to maintain adequate antibody responses to trypanosomes as well as a failure to generate subsequent specific responses to antigens. T. brucei infections in mice result in the loss of mature conventional B cell subsets presumed to be important in host control of the parasites including marginal zone B cells and follicular B cells. Mature cell subset losses are coupled with plasma cell expansion early during infection. Mature B cell pools are not replenished as there is a loss of transitional cells due in part to higher levels of apoptosis and a failure to replenish these cells from bone marrow. B1 B cells appear to constitute the majority of plasma cells and resist infection induced losses to a greater extant than B2 B cells

Distinct in vivo dendritic cell activation by live versus killed Listeria monocytogenes.

Immunization of mice with live or heat-killed Listeria monocytogenes (HKLM) efficiently primes pathogen-specific CD8(+) T cells. T lymphocytes primed by HKLM, however, undergo attenuated proliferation and do not fully differentiate. Thus, only infection with live bacteria induces long-term, CD8(+) T cell-mediated protective immunity. In this study we demonstrate that live and heat-killed bacteria, while both associating with Mac-3(+)CD11b(hi) cells, localize to distinct splenic areas following intravenous inoculation. While HKLM localize to the marginal zone and the splenic red pulp, live L. monocytogenes are carried to the T cell zone of splenic white pulp. Despite these differences, in vivo depletion of CD11c-expressing cells prevents priming of naive T cells by either HKLM or live L. monocytogenes. Analysis of CD11c(hi) dendritic cells (DC) reveals that infection with live L. monocytogenes induces higher levels of CD40, CD80 and CD86 expression than immunization with HKLM. Our results suggest that CD8(+) T cell priming following HKLM immunization or live infection is mediated by DC and that the disparate outcomes of priming can be attributed to suboptimal conditioning of DC in the absence of live, cytosol-invasive bacteria.Comparative StudyJournal ArticleResearch Support, N.I.H. ExtramuralResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.FLWINinfo:eu-repo/semantics/publishe

<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

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

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