CD69+ populations identified by unsupervised FLOCK analyses.

<p>CD3+ CD8+ T_EM events following <i>S</i>. Typhi-infected autologous B-LCL stimulation were uploaded to ImmPort for FLOCK analyses. A) Cytokine/chemokine production patterns of the 6 CD69+ populations identified by FLOCK. B) Histograms showing FLOCK of representative volunteer 53 s at day 10 post immunization with the IL-17A+ populations highlighted (population 1- pink and population 6- aqua).</p

Multifunctional CD8+ T_EM responses to <i>S</i>. Typhi-infected HLA-E restricted cells.

<p>A) Scatter plot showing all combinations of the 6 cytokines/chemokines measured that were positive at one or more time points for representative volunteer 53 s. Increases of >0.5% positive cells over uninfected targets were statistically significant. Each point represents a single time-point and the median value is denoted by a horizontal bar. The cyokine/chemokine combinations with the top 5 median values are indicated by red rectangles. Median values of the subsets of multifunctional cells that show a significant difference (**P<0.01, ***P<0.001), when compared to the subsets without asterisks, are indicated. B) Kinetics of the top 5 populations (as determined by median value) over time.</p

Multifunctional CD8+ T_EM responses to <i>S</i>. Typhi-infected autologous B-LCL.

<p>A) Scatter plot showing all combinations of the 6 cytokines/chemokines measured that were positive at one or more time points for representative volunteer 53 s. Increases of >0.5% positive cells over uninfected targets were statistically significant (P<0.01). Each point represents a single time-point and the median value is denoted by a horizontal bar. The cyokine/chemokine combinations with the top 5 median values are indicated by red rectangles. Median values of the subsets of multifunctional cells that show a significant difference (**P<0.01), when compared to the subsets without asterisks, are indicated. B) Kinetics of the top 5 populations (as determined by median value) over time.</p

Detection of intracellular cytokines/chemokines produced by CD8+ T cell memory subsets in response to stimulation with <i>S.</i> Typhi-infected autologous B-LCL.

<p>Histograms from a representative volunteer (53 s at day 7 post infection) showing the production of 6 cytokines/chemokines by memory T cell subsets following stimulation with <i>S</i>. Typhi-infected autologous B-LCL. CD69 (a marker of recent activation) is displayed on the y-axis and each of the 6 cytokines/chemokines measured are displayed on the x-axes. Column 1 represents total CD8+ T cells, column 2 represents T central memory (T_CM; CD62L+ CD45RA-), column 3 represents T naïve (T_N; CD62L+ CD45RA+), column 4 represents T effector memory (T_EM; CD62L- CD45RA-), and column 5 represents T effector memory CD45RA+ (T_EMRA; CD62L- CD45RA+). The percentage of positive cells is shown for the indicated region in each cytogram.</p

Kinetics of intracellular cytokine/chemokine production following stimulation of PBMC with <i>S</i>. Typhi-infected HLA-E restriced cells.

<p>Intracellular cytokine/chemokine production is shown as the percentage of positive cells. Responses are expressed as net values with day 0 subtracted to normalize for differences in base-line responses to <i>S</i>. Typhi in different volunteers. A) CD8+ T_EM cells for volunteer 53 s B) CD8+ T_EMRA cells for volunteer 53 s C) CD8+ T_EM cells for volunteer 54 s D) CD8+ T_EMRA cells for volunteer 54 s E) CD8+ T_EM cells for volunteer 50 s F) CD8+ T_EMRA cells for volunteer 50 s. Increases of >0.5% cytokine positive cells over uninfected targets were found to be statistically significant (P<0.01). * MIP-1b not measured.</p

Kinetics of intracellular cytokine/chemokine production following stimulation of PBMC with <i>S</i>. Typhi-infected autologous B-LCL.

<p>Intracellular cytokine/chemokine production is shown as the percentage of positive cells. Responses are expressed as net values with day 0 subtracted to normalize for differences in base-line responses to <i>S</i>. Typhi in different volunteers. A) CD8+ T_EM cells for volunteer 53 s B) CD8+ T_EMRA cells for volunteer 53 s C) CD8+ T_EM cells for volunteer 54 s D) CD8+ T_EMRA cells for volunteer 54 s E) CD8+ T_EM cells for volunteer 50 s F) CD8+ T_EMRA cells for volunteer 50 s. Increases of >0.5% cytokine positive cells over uninfected targets were found to be consistently statistically significant (P<0.01) by chi-square analyses. * MIP-1b not measured.</p

Impact of CD4+ T Cell Responses on Clinical Outcome following Oral Administration of Wild-Type Enterotoxigenic <i>Escherichia coli</i> in Humans

<div><p>Enterotoxigenic <i>Escherichia coli</i> (ETEC) is a non-invasive enteric pathogen of considerable public health importance, being one of the most common attributable causes of diarrheal illness in infants and young children in developing countries and the most common cause of traveler’s diarrhea. To enhance study-to-study consistency of our experimental challenge model of ETEC in volunteers, and to allow concomitant multi-site trials to evaluate anti-ETEC immunoprophylactic products, hundreds of vials, each containing a standardized inoculum of virulent wild-type (wt) ETEC strain H10407 (serotype O78:H11 expressing colonization factor antigen I and heat-labile and heat-stable enterotoxins), were prepared under current Good Manufacturing Practices (cGMP) and frozen. Following thawing, the contents of each vial can be used (diluted as necessary) to prepare consistent challenge inoculum, even at different study sites. A preliminary human experimental challenge study using this cGMP inoculum was conducted on a research isolation ward and the clinical and cell-mediated immune responses evaluated. Of the 6 healthy adult volunteers challenged 83% (5/6) developed diarrhea and 50% developed moderate-to-severe diarrhea (MSD). Moderate and severe diarrhea were defined as passage of ≥ 1 liter or ≥ 3 liters of diarrheal stool respectively. We compared the CD4+ T cell responses of volunteers who developed MSD against those who did not and identified significant differences in ETEC-specific cytokine production and gut homing potential. We furthermore demonstrated that increased expression of the gut-homing molecule integrin α4β7 by peripheral T follicular helper cells (pT_fh) correlated with decreased stool volume and increased ETEC-specific IgA B memory cell (B_M) development. Collectively, despite small numbers of volunteers, our results indicate a potential role for CD4+ T cells, in particular pT_fh, in modulating disease outcome following exposure to wt ETEC in a volunteer experimental challenge model.</p></div

Multifunctional CD4+ T cells.

<p>TNF-α and IL-2 multifunctional CD4+ T cells following CFA/I stimulation on day 3 post infection in Resistant volunteers <b>A)</b> 4016 and <b>B)</b> 4001.</p

Individual diarrheal purge to challenge.

<p>Individual diarrheal purge to challenge.</p

Percentage of pT_fh and cytokine production by pT_fh in Resistant and Susceptible volunteers.

<p><b>A)</b> Live single cell CD14- CD19- events were gated on CD3, followed by gating for CD4 and then CXCR5. The percentage of CD4 cells positive for CXCR5 is indicated at baseline (D -7) and 3 days post challenge (D 3). <b>B)</b> Net CFA/I-specific TNF-α production above pre-challenge (D -7) by CD4+ CXCR5+ (pT_fh) on day 3 post-challenge (D3). <b>C)</b> Net CFA/I-specific IL-2 production above pre-challenge (D -7) levels by CD4+ CXCR5+ (pT_fh) on day 3 post-challenge (D3). * p < 0.05 ** p < 0.01</p