Malaria-induced interferon-γ drives the expansion of Tbet^hi atypical memory B cells

<div><p>Many chronic infections, including malaria and HIV, are associated with a large expansion of CD21⁻CD27⁻ ‘atypical’ memory B cells (MBCs) that exhibit reduced B cell receptor (BCR) signaling and effector functions. Little is known about the conditions or transcriptional regulators driving atypical MBC differentiation. Here we show that atypical MBCs in malaria-exposed individuals highly express the transcription factor T-bet, and that T-bet expression correlates inversely with BCR signaling and skews toward IgG3 class switching. Moreover, a longitudinal analysis of a subset of children suggested a correlation between the incidence of febrile malaria and the expansion of T-bet^hi B cells. The Th1-cytokine containing supernatants of malaria-stimulated PBMCs plus BCR cross linking induced T-bet expression in naïve B cells that was abrogated by neutralizing IFN-γ or blocking the IFN-γ receptor on B cells. Accordingly, recombinant IFN-γ plus BCR cross-linking drove T-bet expression in peripheral and tonsillar B cells. Consistent with this, Th1-polarized Tfh (Tfh-1) cells more efficiently induced T-bet expression in naïve B cells. These data provide new insight into the mechanisms underlying atypical MBC differentiation.</p></div

Atypical MBCs and immunoglobulin class switching.

<p>Class-switching of atypical MBCs was assessed by measuring cell surface expression of IgG by flow cytometry in the peripheral blood of <i>Pf</i>-naive U.S. adults (n = 10), <i>Pf</i>-infected Peruvian adults (n = 18) and <i>Pf</i>-infected Malian adults (n = 12). Box-and-whisker plots represent the smallest and largest values (whiskers), the lower and upper quartiles (top and bottom of box), and the median (horizontal line across box). The Mann-Whitney test was used to compare continuous variables between groups.</p

IFN-γ plus BCR crosslinking induce T-bet expression in tonsillar naïve B cells, MBCs and light zone germinal center B cells.

<p>B cell subsets were negatively selected from tonsillar tissue of U.S. children (n = 8) and gated on naïve B cells (CD10-, IgD+), MBCs (CD10-IgD-) and germinal center (GC) (CD10+IgD-) B cells. GC B cells were further stratified on light (CXCR4-) and dark (CXCR4+) zone GC B cells. Gating strategy shown in (<b>A</b>). (<b>B</b>) MFI of total T-bet and <b>(C)</b> percent T-bet^hi determined by FACS in B cell subsets following stimulation with rhIFN-γ, BCR cross-linking or both. p value were determined by 2 way ANOVA with Sidak corrections. ****<i>P</i><0.0001, ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05, ns = not significant.</p

Supernatants of PBMCs stimulated with <i>P</i>. <i>falciparum</i>-infected RBCs plus BCR cross-linking drive T-bet expression in B cells.

<p>(<b>A-C</b>) PBMCs of healthy U.S. adults (n = 5) were stimulated in vitro with <i>P</i>. <i>falciparum</i>-infected red blood cell (iRBC) lysate or uninfected red blood cell (uRBC) lysate for 3 days. The resulting supernatants or the iRBC lysate alone were transferred to PBMCs from the same U.S. adults (n = 5) in the presence of media alone, anti-IgM, anti-CD40, or both, followed by staining for T-bet, CD10, CD19 and IgD. (<b>A</b>) Fold change in T-bet MFI in stimulated naïve B cells relative to unstimulated naïve B cells (left, representative histograms). Fold change in percentage of T-bet intermediate (T-bet^int) and T-bet high (T-bet^hi) (<b>B</b>) naïve B cells and (<b>C</b>) memory B cells after BCR cross-linking with anti-IgM/G/A in the presence of media alone, uRBC/PBMC supernatant or iRBC/PBMC supernatant, relative to unstimulated cells. Horizontal bars and whiskers represent means or median and SE. p values were determined by paired Student’s <i>t</i> test with Bonferroni adjustments where appropriate. ****<i>P</i><0.0001, ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05, ns = not significant.</p

Plasma cytokine response in children during acute malaria.

<p>Plasma cytokine levels in Malian children (n = 37) at their healthy baseline (HB), during acute febrile malaria (Mal) and 7 days after anti-malarial treatment (7dpt). The Th2 cytokines IL-5 and IL-13 were not detectable at any time point. p values were determined by ANOVA with Sidak corrections where appropriate. ****<i>P</i><0.0001, ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05, ns = not significant.</p

Malaria-associated atypical MBCs upregulate <i>TBX21</i>.

<p>(<b>A</b>) Principal components analysis of gene expression of selected regulators of B and T cell differentiation (selected genes shown in <b>B</b>) in naïve B cells (CD19⁺CD21⁺CD27⁻; green), classical MBCs (CD19⁺CD21⁺CD27⁺; pink) and atypical MBCs (CD19⁺CD21⁻CD27⁻; purple). (<b>B</b>) Ex vivo RMA-normalized log2 gene expression values of selected regulators of B and T cell differentiation (rows) for each subject (columns; n = 20) within each B cell subpopulation. Differentially expressed genes in atypical MBCs relative to classical MBCs are indicated with an asterisk.</p

T-bet is highly expressed in malaria-associated atypical MBCs.

<p><b>(A)</b> Ex vivo T-bet expression in total CD19⁺ B cells of representative subjects (right), and Malian children (n = 15) and U.S. adults (n = 10) (left). (<b>B</b>) Ex vivo distribution of T-bet^neg, T-bet^int and T-bet^hi cells stratified by B cell subpopulations in Malian children (n = 15); representative FACS plot (bottom) shows T-bet^neg (black), T-bet^int (blue) and T-bet^hi (purple) cells. p values determined by paired Student’s <i>t</i> test with Bonferroni corrections for multiple comparisons where appropriate. ****<i>P</i><0.0001, ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05, ns = not significant.</p

Atypical MBCs in individuals from the U.S., Peru and Mali.

<p>Atypical MBCs were detected by flow cytometry and expressed as a percentage of total B cells in the peripheral blood of <i>Pf</i>-naive U.S. adults (n = 10), <i>Pf</i>-infected Peruvian adults (n = 18) and <i>Pf</i>-infected Malian adults (n = 12). Box-and-whisker plots represent the smallest and largest values (whiskers), the lower and upper quartiles (top and bottom of box), and the median (horizontal line across box). The Mann-Whitney test was used to compare continuous variables between groups.</p

Change in IgG subclass levels during acute malaria and correlation with plasma IFN-γ levels.

<p>(<b>A</b>) Fold change in total plasma IgG subclasses during acute malaria relative to pre-infection baseline (n = 19 Malian children). (<b>B</b>) Fold change in total plasma IgG subclasses vs fold change in plasma IFN-γ during acute malaria relative to pre-infection baseline (n = 19 Malian children). p values determined by paired Student’s <i>t</i> test with Bonferroni corrections for multiple comparisons where appropriate. Paired Student’s <i>t</i> test and Pearson correlation were used for correlative analyses. ****<i>P</i><0.0001, ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05, ns = not significant.</p

T-bet^hi B cells of malaria-exposed children upregulate the inhibitory receptor FcyR2B.

<p>Ex vivo expression of FcyR2B on total CD19⁺ B cells stratified by level of T-bet expression in Malian children (n = 7; representative subject, right). p values determined by paired Student’s <i>t</i> test with Bonferroni corrections for multiple comparisons where appropriate. ****<i>P</i><0.0001, ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05, ns = not significant.</p