Effector and Central Memory Poly-Functional CD4+ and CD8+ T Cells are Boosted upon ZOSTAVAX® Vaccination

ZOSTAVAX® is a live attenuated varicella-zoster virus (VZV) vaccine that is licensed for the protection of individuals ≥ 50 years against shingles, and its most common complication, post-herpetic neuralgia. While IFN responses increase upon vaccination, the quality of the T cell response has not been elucidated. By using polychromatic flow cytometry, we characterized the breadth, magnitude, and quality of ex vivo CD4+ and CD8+ T cell responses induced 3 – 4 weeks after ZOSTAVAX vaccination of healthy adults. We show, for the first time that the highest frequencies of VZV-specific CD4+ T cells were poly-functional CD154+IFNγ+IL-2+TNFα+ cells, which were boosted upon vaccination. The CD4+ T cells were broadly reactive to several VZV proteins, with IE63 ranking the highest amongst them in the fold-rise of poly-functional cells, followed by IE62, gB, ORF9, and gE. We identified a novel poly-functional ORF9-specific CD8+ T cell population in 62% of the subjects, and these were boosted upon vaccination. Poly-functional CD4+ and CD8+ T cells produced significantly higher levels of IFNγ, IL-2, and TNFα compared to mono-functional cells. After vaccination, a boost in the expression of IFN by poly-functional IE63-and ORF9-specific CD4+ T cells, and IFNγ, IL-2, and TNFα by ORF9-specific poly-functional CD8+ T cells was observed. Responding poly-functional T cells exhibited both effector (CCR7−CD45RA−CD45RO+), and central (CCR7+CD45RA−CD45RO+) memory phenotypes, which expressed comparable levels of cytokines. Altogether, our studies demonstrate that a boost in memory poly-functional CD4+ T cells, and ORF9-specific CD8+ T cells may contribute towards ZOSTAVAX efficacy

A New Type of Proton Coordination in an F1Fo-ATP Synthase Rotor Ring

The high-resolution structure of the rotor ring from alkaliphilic Bacillus pseudofirmus OF4 reveals a new type of ion binding in F1Fo-ATP synthases

Brn3a expression is increased in differentiated iPS cells.

<p>RT-qPCR of Brn3a and GAPDH mRNA was performed. (A) Gel electrophoresis of Brn3a and GAPDH expression in undifferentiated and differentiated iPS cells. The PCR products obtained after 34 cycles was run on a 3% agarose gel. (B) The relative Brn3a gene expression in undifferentiated and differentiated iPS cells (unpaired t test, p<0.0004). Data shown are mean ± SEM of three biological replicates.</p

Neurons derived from iPS cells support action potentials.

<p>Whole-cell patch-clamp recordings were made in current-clamp mode from the soma of neurons. Shown above are representative traces recorded from one neuron that generated action potentials in response to depolarization. The stimulus protocol is depicted in lower traces. Membrane potentials were recorded from four neurons.</p

HSV infects iPS cells at all stages of differentiation.

<p>Cells were infected with cell-free HSV at a MOI of 0.1 for 96 hours. (A) DAPI staining identifies the iPS cell colonies (highlighted by the dotted white lines). (B) Immunostaining for gD revealed that both iPS cells and the underlying fibroblasts supported HSV infection, as evidenced by abundant gD expression. (C) Merge of panels A and B. (D–F) Sensory neurons derived from iPS cells also supported HSV infection. Staining for gD (D) and Brn3a (E). (F) Merge of panels D and E. Scale for all images is 100 um.</p

Differentiation of neural progenitor cells to sensory neurons.

<p>(A) Brightfield images of neural progenitor cells on days 2, 8 and 14 in media containing growth factors. (B) Twenty-four days after initiating differentiation of iPS cells, neurons expressed the neuronal marker βIII-tubulin. Nuclei were visualized with DAPI. (C) Approximately 80% of differentiated iPS cells are βIII-tubulin+ as determined by flow cytometry. The red tracing represents unstained cells and the blue tracing represents cells stained with an antibody to βIII-tubulin. (D) Cells also expressed peripherin and Brn3a, markers of sensory neurons. (E) The percentage of peripherin+, Brn3a+, and peripherin+/Brn3a+ cells was determined by flow cytometry. Data shown are the mean ± SEM. Co-expression of Islet-1 with (F) Brn3a and (G) peripherin. Scale for all images is 100 um.</p

Conversion of human iPS cells to neural progenitor cells.

<p>(A) Outline of the differentiation protocol. Human iPS cells were dissociated and plated on Matrigel-coated plates. For the first 10 days, they were exposed to small molecule inhibitors, followed by culturing for two weeks in growth factors. (B) Brightfield images of iPS cells after 4 and 10 days of exposure to small molecule inhibitors. After 10 days, iPS cells expressed (C) Pax6 and (D) nestin, markers of neural progenitor cells. Nuclei were visualized with DAPI. Scale for all images is 100 um.</p