CD4⁺ T cells from protected Subject A produce proinflammatory cytokines following infection with live <i>C. jejuni</i>.

<p>PBMCs from pre-challenge (D0) and post-infection timeponts were stimulated with <i>C. jejuni</i> antigen in an <i>ex vivo</i> assay and assessed for CD4⁺ cytokine production by flow cytometry. Responses depicted represent the percentage of CD4⁺ T cells producing cytokine from CAg-stimulated PBMCs (CAg stimulation minus negative control). (*150 =  Day of homologous re-challenge.)</p

Proinflammatory cytokines and a chemokine are produced with specific kinetics post-primary infection with <i>C. jejuni</i>.

<p>We analyzed CD4⁺ T cells for the production of IFNγ⁺, TNFα⁺, IL-2⁺, and MIP-1β⁺ for eight subjects challenged with <i>C. jejuni</i> at multiple timepoints pre- and post-primary infection. Responses shown are the percentage of cytokine positive CD4⁺ T cells from CAg-stimulated PBMCs with the background percentage of cytokine-positive T cells in the negative control (PBS) subtracted. CD4⁺ T cells producing each cytokine were reported for 3 time-points post-challenge: D7 (blue), D14 (red), and D28 (black). A) CD4⁺IFNγ⁺ T cells; B) CD4⁺TNFα⁺ T cells; C) CD4⁺IL-2⁺ T cells; and D) CD4⁺MIP-1β⁺ T cells. An asterisk denotes a significant difference between a response on a post-challenge day and D0 (yellow): * P<.05 and ** P<.01. Statistics were determined using repeated measures analysis of variance were not available for Subject B after re-infection.</p

CD4⁺ T cell responses did not show a specific signature following re-challenge with the homologous <i>C. jejuni</i> strain.

<p>The effect of challenge on <i>C. jejuni</i> veterans shows a diverse T cell profile compared to naïve subjects. Eight subjects were re-dosed with the homologous <i>C. jejuni</i> CG8421 strain three months after initial infection. We examined CD4⁺ T cell responses for the production of IFNγ⁺, TNFα⁺, IL-2⁺, and MIP-1β at multiple timepoints pre- and post-re-infection. Responses shown are the percentage of cytokine positive CD4⁺ T cells from CAg-stimulated PBMCs with the background percentage of cytokine-positive T cells in the negative control (PBS) subtracted. D98 represents PBMCs from a blood drawn prior to re-infection. Bars represent percent of CD4⁺ T cells detected on specific days: D98 (day of re-dosing, yellow); D105 (blue); D112 (red); and D126 (black). No significant changes were observed post re-challenge compared to D98.</p

Peripheral CD4⁺ T Cell Cytokine Responses Following Human Challenge and Re-Challenge with <i>Campylobacter jejuni</i>

<div><p><i>Campylobacter jejuni</i> is a leading cause of human gastroenteritis worldwide; however, our understanding of the human immune response to <i>C. jejuni</i> infection is limited. A previous human challenge model has shown that <i>C. jejuni</i> elicits IFNγ production by peripheral blood mononuclear cells, a response associated with protection from clinical disease following re-infection. In this study, we investigate T lymphocyte profiles associated with campylobacteriosis using specimens from a new human challenge model in which <i>C. jejuni</i>-naïve subjects were challenged and re-challenged with <i>C. jejuni</i> CG8421. Multiparameter flow cytometry was used to investigate T lymphocytes as a source of cytokines, including IFNγ, and to identify cytokine patterns associated with either campylobacteriosis or protection from disease. Unexpectedly, all but one subject evaluated re-experienced campylobacteriosis after re-challenge. We show that CD4⁺ T cells make IFNγ and other pro-inflammatory cytokines in response to infection; however, multifunctional cytokine response patterns were not found. Cytokine production from peripheral CD4⁺ T cells was not enhanced following re-challenge, which may suggest deletion or tolerance. Evaluation of alternative paradigms or models is needed to better understand the immune components of protection from campylobacteriosis.</p></div

Monofunctional CD4⁺ T cells from naïve and veterans post <i>C. jejuni</i> exposure are dominant over multifunctional cells.

<p>The kinetics of T cell responses are shown, separating CD4⁺ phenotypes based on the combinations of cytokines they produced (designated by +/-) under the x-axis. X-axis shows the combinations of IFNγ, TNFα, IL-2, and MIP-1β producing cells. Each colored bar designates the interquartile range (IQR). Days evaluated include: D0, D7, D14, D28, D98, D105, D112, and D126. A) Monofunctional CD4⁺ phenotypes. B and C) Multifunctional CD4⁺ phenotypes relative to D0. Statistically significant days are relative to D0 (Wilcoxon ranked test) and P≤0.05 is designated by “<b>*</b>”.</p

Primary Vaccination with Low Dose Live Dengue 1 Virus Generates a Proinflammatory, Multifunctional T Cell Response in Humans

<div><p>The four dengue virus serotypes (DENV-1–DENV-4) have a large impact on global health, causing 50–100 million cases of dengue fever annually. Herein, we describe the first kinetic T cell response to a low-dose DENV-1 vaccination study (10 PFU) in humans. Using flow cytometry, we found that proinflammatory cytokines, IFNγ, TNFα, and IL-2, were generated by DENV-1-specific CD4⁺ cells 21 days post-DENV-1 exposure, and their production continued through the latest time-point, day 42 (<em>p</em><0.0001 for all cytokines). No statistically significant changes were observed at any time-points for IL-10 (<em>p</em> = 0.19), a regulatory cytokine, indicating that the response to DENV-1 was primarily proinflammatory in nature. We also observed little T cell cross-reactivity to the other 3 DENV serotypes. The percentage of multifunctional T cells (T cells making ≥2 cytokines simultaneously) increased with time post-DENV-1 exposure (<em>p</em><0.0001). The presence of multifunctional T cells together with neutralizing antibody data suggest that the immune response generated to the vaccine may be protective. This work provides an initial framework for defining primary T cell responses to each DENV serotype and will enhance the evaluation of a tetravalent DENV vaccine.</p> </div

Proinflammatory cytokines are produced at specific times post-vaccination with live DENV-1.

<p>Cytokine profiles for 12 subjects vaccinated with 10 PFU of live monovalent DENV-1 and 2 placebo recipients. CD4⁺-dependent production of 4 cytokines are shown for pre-vaccination (day 0) and 5 post-vaccination time-points following <i>ex vivo</i> stimulation with homologous, wild type DENV-1 antigen: <b>A.</b> IFNγ, <b>B.</b> TNFα, <b>C.</b> IL-2 and <b>D.</b> IL-10. Responses shown are the percentage of cytokine positive T cells from DENV-1-stimulated PBMCs with the background percentage of cytokine-positive T cells in the negative control (Vero) subtracted. Clinical information for each subject is shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001742#pntd-0001742-t001" target="_blank"><b>Table 1</b></a>. Significantly higher levels of IFNγ and TNFα were observed on days 21, 28 and 42 (<i>p</i><0.05 for IFNγ and <i>p</i><0.01 for TNFα), and on days 21 and 28 for IL-2 (<i>p</i><0.01). For <b>A–C</b>, <i>p</i><0.0001 for group by day differences demonstrating increased cytokines with time compared to day 0.</p

Little T cell crossreactivity when stimulated with heterologous DENV antigens.

<p>Cytokine production in CD4⁺ T cells for 2 subjects (Subject 06, viremic (<b>A–D</b>) and Subject 08, non-viremic (<b>E–H</b>)) in response to stimulation with homologous (DENV-1, black bars) or heterologous stimulation with DENV-2 (yellow), DENV-3 (blue) or DENV-4 (red). Shown are the % CD4⁺ T cells producing a particular cytokine at pre-vaccination (day 0) and post-vaccination time-points: IFNγ (<b>A, E</b>), TNFα (<b>B, F</b>), IL-2 (<b>C, G</b>), and IL-10 (<b>D, H</b>). Insufficient cells were available at day 42 for <b>E</b>, <b>F</b>, <b>G</b>, <b>H</b> for analysis.</p

DENV-1 vaccinees display a variety of clinical signs, symptoms and immunologic data.

a<p>Historical data as described in (Lindow, unpublished).</p>b<p>Viremia was 0.5 log₁₀/ml in serum on the days indicated as previously described (Lindow, unpublished).</p>c<p>PRNT₆₀ indicates the antibody titer required to neutralize 60% of wild type DENV-1 virus relative to a no serum control.</p

Multifunctional T cells are produced following exposure to 10 PFU of a DENV-1 monovalent vaccine.

<p><b>A.</b> Distribution of cytokines produced by multifunctional or monofunctional T cells. The percent of multifunctional CD4⁺ T cells producing IFNγ (black line), TNFα (black dashes), and/or IL-2 (black dots) over time relative to the number of CD4⁺ T cells making a single cytokine: IFNγ (gray line), TNFα (gray dashes) or IL-2 (gray dots). Error bars represent standard error, n = 9–11 depending on time-point. <b>B.</b> IFNγ, IL2, and TNFα production in CD4⁺ T cells as a function of time and co-production. Each day is represented with a single color. Black dots correspond to the response from a single subject. Shaded bars represent the interquartile range. Line separates multifunctional T cells (left) from monofunctional T cells (right). A “+” or “−” indicates whether a particular cytokine (TNFα, IFNγ and/or IL-2) is present. <b>C.</b> Proportions of total CD4⁺ T cells producing IFNγ (blue), IL2 (green), TNFα (red), or different combinations of the three (colors indicated by boxes in <b>B</b>) are shown for 5 post-vaccination time-points (days 8, 14, 21, 28, and 42) relative to pre-vaccination (day 0) for 11 vaccinees. Overlaps in red, green, and/or blue arcs indicate T cells producing the correlated cytokines simultaneously. A “ #” denotes statistically a significant difference between a response on a post-vaccination day and day 0 (<i>p</i><0.05 by Wilcoxon's rank sum test). There are statistically higher percentages of total multifunctional T cells on days 21, 28, and 42 (<i>p</i><0.0001).</p