Limits...
Plasmodium chabaudi infection induces AID expression in transitional and marginal zone B cells

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Endemic Burkitt's lymphoma (eBL) is associated with Epstein–Barr virus and repeated malaria infections. A defining feature of eBL is the translocation of the c‐myc oncogene to the control of the immunoglobulin promoter. Activation‐induced cytidine deaminase (AID) has been shown to be critical for this translocation. Malaria infection induces AID in germinal center B cells, but whether malaria infection more broadly affects AID activation in extrafollicular B cells is unknown.

Methods: We either stimulated purified B cells from AID‐green fluorescence protein (GFP) reporter mice or infected AID‐GFP mice with Plasmodium chabaudi, AID fluorescence was monitored in B cell subsets by flow cytometry.

Results: In vitro analysis of B cells from these mice revealed that CpG (a Toll‐like receptor 9 ligand) was a potent inducer of AID in both mature and immature B cell subsets. Infection of AID‐GFP mice with Plasmodium chabaudi demonstrated that AID expression occurs in transitional and marginal zone B cells during acute malaria infection. Transitional B cells were also capable of differentiating into antibody secreting cells when stimulated in vitro with CpG when isolated from a P. chabaudi‐infected mouse.

Conclusions: These data suggest that P. chabaudi is capable of inducing AID expression in B cell subsets that do not participate in the germinal center reaction, suggesting an alternative role for malaria in the etiology of eBL.

No MeSH data available.


Related in: MedlinePlus

CpG stimulation leads to AID expression in mature, and T1 B cells and CpG+anti‐IgM resulted in the expression of AID in T2 B cells. B cells enriched by negative selection from AID‐GFP mice were cultured in the presence of media alone, CpG, or CpG+anti‐IgM for 3 days. Populations of mature, T1 and T2 B cell subsets were analyzed for their expression of GFP. Representative flow cytometry showing the mature (CD19+CD93−) and immature (CD19+CD93+) B cells on the left panel. Transitional types 1 and 2 B cells were differentiated in the second column by IgM and CD23 expression (T1 = IgM+, CD23− and T2 = IgM+, CD23 +). The right three columns show AID‐GFP expression for the mature B cells (CD19+ CD93−), T1 cells, and T2 cells compared to a cell proliferation dye (n = 3 in two independent experiments).
© Copyright Policy - creativeCommonsBy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5134720&req=5

iid3134-fig-0001: CpG stimulation leads to AID expression in mature, and T1 B cells and CpG+anti‐IgM resulted in the expression of AID in T2 B cells. B cells enriched by negative selection from AID‐GFP mice were cultured in the presence of media alone, CpG, or CpG+anti‐IgM for 3 days. Populations of mature, T1 and T2 B cell subsets were analyzed for their expression of GFP. Representative flow cytometry showing the mature (CD19+CD93−) and immature (CD19+CD93+) B cells on the left panel. Transitional types 1 and 2 B cells were differentiated in the second column by IgM and CD23 expression (T1 = IgM+, CD23− and T2 = IgM+, CD23 +). The right three columns show AID‐GFP expression for the mature B cells (CD19+ CD93−), T1 cells, and T2 cells compared to a cell proliferation dye (n = 3 in two independent experiments).

Mentions: In vitro studies of human immature B cells have shown that CpG stimulation through TLR9 signaling is capable of inducing AID activity 10. We wanted to determine whether TLR9 signaling in B cells induces AID in a mouse model system using an AID‐GFP transgenic reporter mouse 18. To do this, we enriched splenic B cells by negative selection. Following B cell enrichment, we obtained a heterogeneous population of B cell subsets that included both immature and mature B cells (CD19 + CD93+ and CD19 + CD93−, respectively). We then stained the cells with a proliferation dye stimulated these B cells with CpG and anti‐IgM F(ab′)2 fragments to determine whether CpG alone, or in conjunction with BCR crosslinking, activated AID expression. AID expression was then measured in immature and mature B cell subsets after 3 days stimulation by flow cytometry. When cultured in the absence of stimulation the immature B cells (CD19+, CD93+) made up <1% of total cells in as little as 1 day in culture (data not shown) and remained absent after 3 days (Fig. 1). Additionally, unstimulated B cells expressed low levels of CD19 and were almost completely AID negative (Fig. 1). Stimulation with anti‐IgM alone resulted in the same phenotype as control cells for all parameters analyzed (data not shown). After 3 days in culture, CpG‐stimulated B cells comprised two populations of CD19high and CD19low cells. The CD19high population consisted of the blasting cells that had undergone division and included both immature and mature B cells (Fig. 1). The majority of the CpG‐stimulated immature B cells were AID+ and had an IgM+CD23− or transitional type 1 (T1) phenotype (Fig. 1). When CpG stimulation was combined with BCR cross‐linking with anti‐IgM, there were similar levels of AID expression as CpG alone. Interestingly, the phenotype of the immature B cells following treatment with both anti‐IgM and CpG included both T1 and IgM+ CD23+ transitional type 2 (T2) B cells (Fig. 1). These data demonstrate that CpG has the ability to induce AID expression in vitro in both mature and T1 B cells, and that anti‐IgM treatment in conjunction with CpG is capable of leading to persistence of AID expressing T2 B cells.


Plasmodium chabaudi infection induces AID expression in transitional and marginal zone B cells
CpG stimulation leads to AID expression in mature, and T1 B cells and CpG+anti‐IgM resulted in the expression of AID in T2 B cells. B cells enriched by negative selection from AID‐GFP mice were cultured in the presence of media alone, CpG, or CpG+anti‐IgM for 3 days. Populations of mature, T1 and T2 B cell subsets were analyzed for their expression of GFP. Representative flow cytometry showing the mature (CD19+CD93−) and immature (CD19+CD93+) B cells on the left panel. Transitional types 1 and 2 B cells were differentiated in the second column by IgM and CD23 expression (T1 = IgM+, CD23− and T2 = IgM+, CD23 +). The right three columns show AID‐GFP expression for the mature B cells (CD19+ CD93−), T1 cells, and T2 cells compared to a cell proliferation dye (n = 3 in two independent experiments).
© Copyright Policy - creativeCommonsBy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5134720&req=5

iid3134-fig-0001: CpG stimulation leads to AID expression in mature, and T1 B cells and CpG+anti‐IgM resulted in the expression of AID in T2 B cells. B cells enriched by negative selection from AID‐GFP mice were cultured in the presence of media alone, CpG, or CpG+anti‐IgM for 3 days. Populations of mature, T1 and T2 B cell subsets were analyzed for their expression of GFP. Representative flow cytometry showing the mature (CD19+CD93−) and immature (CD19+CD93+) B cells on the left panel. Transitional types 1 and 2 B cells were differentiated in the second column by IgM and CD23 expression (T1 = IgM+, CD23− and T2 = IgM+, CD23 +). The right three columns show AID‐GFP expression for the mature B cells (CD19+ CD93−), T1 cells, and T2 cells compared to a cell proliferation dye (n = 3 in two independent experiments).
Mentions: In vitro studies of human immature B cells have shown that CpG stimulation through TLR9 signaling is capable of inducing AID activity 10. We wanted to determine whether TLR9 signaling in B cells induces AID in a mouse model system using an AID‐GFP transgenic reporter mouse 18. To do this, we enriched splenic B cells by negative selection. Following B cell enrichment, we obtained a heterogeneous population of B cell subsets that included both immature and mature B cells (CD19 + CD93+ and CD19 + CD93−, respectively). We then stained the cells with a proliferation dye stimulated these B cells with CpG and anti‐IgM F(ab′)2 fragments to determine whether CpG alone, or in conjunction with BCR crosslinking, activated AID expression. AID expression was then measured in immature and mature B cell subsets after 3 days stimulation by flow cytometry. When cultured in the absence of stimulation the immature B cells (CD19+, CD93+) made up <1% of total cells in as little as 1 day in culture (data not shown) and remained absent after 3 days (Fig. 1). Additionally, unstimulated B cells expressed low levels of CD19 and were almost completely AID negative (Fig. 1). Stimulation with anti‐IgM alone resulted in the same phenotype as control cells for all parameters analyzed (data not shown). After 3 days in culture, CpG‐stimulated B cells comprised two populations of CD19high and CD19low cells. The CD19high population consisted of the blasting cells that had undergone division and included both immature and mature B cells (Fig. 1). The majority of the CpG‐stimulated immature B cells were AID+ and had an IgM+CD23− or transitional type 1 (T1) phenotype (Fig. 1). When CpG stimulation was combined with BCR cross‐linking with anti‐IgM, there were similar levels of AID expression as CpG alone. Interestingly, the phenotype of the immature B cells following treatment with both anti‐IgM and CpG included both T1 and IgM+ CD23+ transitional type 2 (T2) B cells (Fig. 1). These data demonstrate that CpG has the ability to induce AID expression in vitro in both mature and T1 B cells, and that anti‐IgM treatment in conjunction with CpG is capable of leading to persistence of AID expressing T2 B cells.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Endemic Burkitt's lymphoma (eBL) is associated with Epstein&ndash;Barr virus and repeated malaria infections. A defining feature of eBL is the translocation of the c&#8208;myc oncogene to the control of the immunoglobulin promoter. Activation&#8208;induced cytidine deaminase (AID) has been shown to be critical for this translocation. Malaria infection induces AID in germinal center B cells, but whether malaria infection more broadly affects AID activation in extrafollicular B cells is unknown.

Methods: We either stimulated purified B cells from AID&#8208;green fluorescence protein (GFP) reporter mice or infected AID&#8208;GFP mice with Plasmodium chabaudi, AID fluorescence was monitored in B cell subsets by flow cytometry.

Results: In vitro analysis of B cells from these mice revealed that CpG (a Toll&#8208;like receptor 9 ligand) was a potent inducer of AID in both mature and immature B cell subsets. Infection of AID&#8208;GFP mice with Plasmodium chabaudi demonstrated that AID expression occurs in transitional and marginal zone B cells during acute malaria infection. Transitional B cells were also capable of differentiating into antibody secreting cells when stimulated in vitro with CpG when isolated from a P. chabaudi&#8208;infected mouse.

Conclusions: These data suggest that P. chabaudi is capable of inducing AID expression in B cell subsets that do not participate in the germinal center reaction, suggesting an alternative role for malaria in the etiology of eBL.

No MeSH data available.


Related in: MedlinePlus