B cell subset analysis of HIV+ (n = 14) and HIV- (n = 21) subjects at the time of symptomatic malaria.

<p>The B cell subsets were determined by flow cytometry: naïve cells (CD19+CD10-CD21+CD27-), activated MBCs/plasmablasts (CD19+CD10-CD21-CD27+), classical MBCs (CD19+CD10-CD21+CD27+) and atypical MBCs (CD19+CD10-CD21-CD27-). The black bar denotes median values. The frequency was determined as percent of total CD19+ B cells. The Mann Whitney rank-sum test was used to compare variables between groups.</p

Demographics and clinical characteristics of the HIV positive (HIV+) and HIV negative (HIV-) malaria infected patients.

<p>P-values were generated using Mann-Whitney test for continuous variables and the Chi-square test for gender. Median and interquartile values are reported.</p><p>Demographics and clinical characteristics of the HIV positive (HIV+) and HIV negative (HIV-) malaria infected patients.</p

Comparison of antibody breadth and magnitude between HIV+ and HIV- samples for the P. falciparum antigens displaying the greatest breadth of antibody reactivity in HIV+ samples.

<p>Frequency of detection in percent of samples by HIV status is reported. Significant differences in breadth are denoted by *, using Fisher exact test (two tailed, p value <0.05). Significant differences in antibody magnitude are denoted by **, and reported using the Empirical Bayes Moderated t-test, p<0.05, and an absolute log fold change > 1.</p><p>Comparison of antibody breadth and magnitude between HIV+ and HIV- samples for the P. falciparum antigens displaying the greatest breadth of antibody reactivity in HIV+ samples.</p

Breadth and magnitude of the IgG response to <i>P</i>. <i>falciparum</i> antigens by HIV status.

<p>(A) A microarray containing 824 <i>P</i>. <i>falciparum</i> proteins or protein fragments was probed with plasma samples from HIV+ (n = 18) and HIV- (n = 18) adults during symptomatic malaria. A. Venn diagrams showing the number of reactive antigens among HIV+ subjects (orange), HIV- subjects (blue), both HIV+ and HIV- subjects (purple) or neither (254). (B) Antibody breadth of HIV+ individuals (mean 83 antigens) and HIV- individuals (mean 208 antigens). Mean values and standard deviations are shown; Significant differences in breadth (Negative Binomial generalized linear model) (C) Magnitude of <i>P</i>. <i>falciparum</i> IgG responses by HIV status. We examined 384 antigens that were recognized in ≥ 10% of all samples and show the average IgG reactivity of each by HIV status. IgG reactivity is significantly higher in HIV- group (blue bars) compared to HIV+ group (orange bars) for 173 antigens. The red horizontal line indicates a p value of 0.05. (Empirical Bayes Moderated t-test, p<0.05, and an absolute log fold change > 1).</p

Number of reactive antibodies per sample in the HIV+ group by HIV viral load and CD4+ T cell count.

<p>The four samples with the highest antibody breadth all have CD4⁺ T cell counts >500 cells/μl and low viral loads. Dotted line denotes HIV viral load limit of detection.</p

Kinetics of vaccine-specific IgG levels in children during the dry season and after acute malaria.

<p><b>(A)</b> The study was designed to take advantage of the sharply demarcated and intense 6-month malaria season (July—December) and 6-month dry season (January—June; negligible malaria transmission) in Mali. Shown is the number of febrile malaria episodes per day over two years at the study site in a cohort of 695 children and adults. <b>(B)</b> IgG levels specific for routine vaccines administered under one year of age (tetanus, measles and Hepatitis B) were measured in plasma collected from 54 children at four time points (vertical arrows): before and after the 6-month dry season, 10 days after the first acute malaria episode of the ensuing malaria season, and after the second dry season. Shown for each subject are IgG titers specific for <b>(C)</b> tetanus, <b>(D)</b> measles and <b>(E)</b> hepatitis B vaccines at the time points indicated in <b>(B)</b>. The x-axis indicates the age at which the respective time points occurred for each subject. In red are subjects who experienced an accelerated decline in vaccine-specific IgG titers following acute malaria (between 2^nd and 3^rd time points) relative to each child’s own rate of change during the preceding dry season (between 1^st and 2^nd time points). The percentage of subjects for whom malaria was associated with an accelerated decline in IgG is shown in red text for each vaccine. A linear mixed effects model that included three time points over 18 months (before and after the first dry season, and after the second dry season) was used to estimate average IgG half-lives for all subjects (black dashed line) and separately for children aged ≤3 years (green dotted line) and >3 years of age (blue dash-dot line).</p

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

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

Rate of change of vaccine-specific IgG levels during the dry season versus during malaria exposure and percentage of children with protective IgG levels.

<p>¹Results based on the last time point of the study period.</p><p>²Standard error.</p><p>³p value for the difference in slopes between the dry season and period of malaria exposure was obtained by fitting a linear mixed effects model.</p><p>⁴Indicates that the slope is significantly different than zero.</p><p>Rate of change of vaccine-specific IgG levels during the dry season versus during malaria exposure and percentage of children with protective IgG levels.</p

Demographic and clinical characteristics of study subjects.

<p>¹Interquartile range.</p><p>²Malaria episode defined as T≥37.5°C, asexual parasitemia ≥2500/microliter and no other cause of fever discernible on physical examination.</p><p>³Days since enrollment during a cross-sectional survey before the malaria season in May 2012.</p><p>⁴95% confidence interval.</p><p>Demographic and clinical characteristics of study subjects.</p