Investigating and Manipulating Immune Responses to Hepatotropic Pathogens Using Synthetic DNA

Hepatotropic pathogens, such as Hepatitis B virus (HBV), Hepatitis C virus (HCV) and malaria Plasmodium often escape cellular immune clearance, resulting in chronic infections. With billions at the risk of infection, the need for an immune therapy that will incite protective immune responses against these pathogens is more important now than ever. To develop effective therapies against these pathogens, it is important to understand the mechanisms by which liver-primed CD8 T cells become defective. In this report, I directly compared liver-primed CD8 T cells to secondary lymphoid tissue-primed CD8 T cells for differentiation, function, and memory programming in a highly controlled fashion. We used hydrodynamic tail vain injection of synthetic plasmids to establish liver-specific antigen expression in the P14 transgenic mouse model, and studied the priming of CD8 T cells. Intrahepatically activated CD8 T cells exhibited unique expansion, memory differentiation, polyfunctionality and cytotoxicity compared to T cells primed in the periphery. The difference in their expansion resulted in lower memory CTL frequency, which led to reduced protection against lethal viral challenge. These results demonstrated that defective liver priming of naïve CD8 T cells contributes to the lower frequency of antigen-specific CTLs observed during liver infection in HBV and HCV patients, which helps these pathogens to escape immune clearance. The results from the study provide evidence that, the eradication of HBV and HCV infected hepatocytes will require both the induction of a strong antigen-specific immune response and the subsequent deployment of that response towards the liver. We therefore assessed the ability of a synthetic DNA vaccine encoding a recombinant plasmid of the HBcAg and HBsAg to drive immunity in the liver. Intramuscular vaccination accompanied by electroporation induced both strong antigen-specific T cell and high titer antibody responses systematically and in the liver. Furthermore, immunized mice showed strong cytotoxic responses that eliminate adoptively transferred HBV-coated target cells in the spleen and liver. These data provide important insight into the generation of peripheral immune responses that are recruited to the liver; an approach that could be beneficial in the search for vaccines or immune-therapies for liver disease

PD-1 Co-inhibitory and OX40 Co-stimulatory Crosstalk Regulates Helper T Cell Differentiation and Anti-Plasmodium Humoral Immunity

SummaryThe differentiation and protective capacity of Plasmodium-specific T cells are regulated by both positive and negative signals during malaria, but the molecular and cellular details remain poorly defined. Here we show that malaria patients and Plasmodium-infected rodents exhibit atypical expression of the co-stimulatory receptor OX40 on CD4 T cells and that therapeutic enhancement of OX40 signaling enhances helper CD4 T cell activity, humoral immunity, and parasite clearance in rodents. However, these beneficial effects of OX40 signaling are abrogated following coordinate blockade of PD-1 co-inhibitory pathways, which are also upregulated during malaria and associated with elevated parasitemia. Co-administration of biologics blocking PD-1 and promoting OX40 signaling induces excessive interferon-gamma that directly limits helper T cell-mediated support of humoral immunity and decreases parasite control. Our results show that targeting OX40 can enhance Plasmodium control and that crosstalk between co-inhibitory and co-stimulatory pathways in pathogen-specific CD4 T cells can impact pathogen clearance

Circulating Th1-Cell-type Tfh Cells that Exhibit Impaired B Cell Help Are Preferentially Activated during Acute Malaria in Children

Malaria-specific antibody responses are short lived in children, leaving them susceptible to repeated bouts of febrile malaria. The cellular and molecular mechanisms underlying this apparent immune deficiency are poorly understood. Recently, T follicular helper (Tfh) cells have been shown to play a critical role in generating long-lived antibody responses. We show that Malian children have resting PD-1+CXCR5+CD4+ Tfh cells in circulation that resemble germinal center Tfh cells phenotypically and functionally. Within this population, PD-1+CXCR5+CXCR3− Tfh cells are superior to Th1-polarized PD-1+CXCR5+CXCR3+ Tfh cells in helping B cells. Longitudinally, we observed that malaria drives Th1 cytokine responses, and accordingly, the less-functional Th1-polarized Tfh subset was preferentially activated and its activation did not correlate with antibody responses. These data provide insights into the Tfh cell biology underlying suboptimal antibody responses to malaria in children and suggest that vaccine strategies that promote CXCR3− Tfh cell responses may improve malaria vaccine efficacy

Longitudinal analysis of naturally acquired PfEMP1 CIDR domain variant antibodies identifies associations with malaria protection

BACKGROUND Malaria pathogenicity is determined, in part, by the adherence of Plasmodium falciparum–infected erythrocytes to the microvasculature mediated via specific interactions between P. falciparum erythrocyte membrane protein (PfEMP1) variant domains and host endothelial receptors. Naturally acquired antibodies against specific PfEMP1 variants can play an important role in clinical protection against malaria. METHODS We evaluated IgG responses against a repertoire of PfEMP1 CIDR domain variants to determine the rate and order of variant-specific antibody acquisition and their association with protection against febrile malaria in a prospective cohort study conducted in an area of intense, seasonal malaria transmission. RESULTS Using longitudinal data, we found that IgG antibodies against the pathogenic domain variants CIDRα1.7 and CIDRα1.8 were acquired the earliest. Furthermore, IgG antibodies against CIDRγ3 were associated with reduced prospective risk of febrile malaria and recurrent malaria episodes. CONCLUSION This study provides evidence that acquisition of IgG antibodies against PfEMP1 variants is ordered and demonstrates that antibodies against CIDRα1 domains are acquired the earliest in children residing in an area of intense, seasonal malaria transmission. Future studies will need to validate these findings in other transmission settings and determine the functional activity of these naturally acquired CIDR variant–specific antibodies. TRIAL REGISTRATION ClinicalTrials.gov NCT01322581

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

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

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

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