QUANTITATIVE ANALYSIS OF SHAPE VARIATION IN TYPE AND MODERN POPULATIONS OF MERIDION (BACILLARIOPHYCEAE)

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65187/1/j.1529-8817.2001.037001175.x.pd

Multiple Dendritic Cell Populations Activate CD4+ T Cells after Viral Stimulation

Dendritic cells (DC) are a heterogeneous cell population that bridge the innate and adaptive immune systems. CD8α DC play a prominent, and sometimes exclusive, role in driving amplification of CD8+ T cells during a viral infection. Whether this reliance on a single subset of DC also applies for CD4+ T cell activation is unknown. We used a direct ex vivo antigen presentation assay to probe the capacity of flow cytometrically purified DC populations to drive amplification of CD4+ and CD8+ T cells following infection with influenza virus by different routes. This study examined the contributions of non-CD8α DC populations in the amplification of CD8+ and CD4+ T cells in cutaneous and systemic influenza viral infections. We confirmed that in vivo, effective immune responses for CD8+ T cells are dominated by presentation of antigen by CD8α DC but can involve non-CD8α DC. In contrast, CD4+ T cell responses relied more heavily on the contributions of dermal DC migrating from peripheral lymphoid tissues following cutaneous infection, and CD4 DC in the spleen after systemic infection. CD4+ T cell priming by DC subsets that is dependent upon the route of administration raises the possibility that vaccination approaches could be tailored to prime helper T cell immunity

Multiple subsets of DC present influenza HA antigen to T cells in BALB/c mice infected with PR8 virus by s.c. injection into the footpad.

<p>The popliteal lymph nodes were isolated and non-DC lineages were removed by antibody depletion. CD11c⁺ DC subsets were purified by flow cytometric sorting into DN DC, CD8α DC and pDC before culturing titrating numbers of each subset with 5×10⁴ CFSE-labelled influenza-specific CD8⁺ T cells (<i>A,C</i>) or CD4⁺ T cells (<i>B,D</i>) 48 (<i>A,B</i>) and 72 (<i>C,D</i>) h after infection. The x-axis shows the range of DC used as stimulators over different experiments. Proliferation was analysed at 60 h of culture. Data are pooled from (<i>A–C</i>) three and four (<i>D</i>) independent experiments and show the mean and SEM. Significant differences (<i>p</i>≤0.05) in proliferation induced by different DC populations are indicated by an asterisk.</p

DC were isolated from the popliteal lymph node or spleen of mice and flow cytometrically purified into DC subsets according to one of three sorting protocols.

<p><i>A</i>. In the first approach, CD11c⁺ cells were sorted from the popliteal lymph node or spleen into CD8α⁺ (CD8α) DC, CD8α^+/−CD45RA⁺ plasmacytoid DC (pDC) and CD8α⁻CD45RA⁻ double negative (DN) DC. <i>B.</i> In the second approach, CD11c⁺ cells from popliteal lymph node were further dissected into CD8⁻CD205^high Langerhans cells, CD8α⁻CD205^int dermal DC, CD8α⁻CD205⁻ DN DC and CD8α⁺CD205^int DC. <i>C</i>. In the third approach, CD11c⁺ cells were sorted from light density cells isolated from spleen into CD8α⁺CD4⁻, CD8α⁻CD4⁺ and CD8α⁻CD4⁻ DC.</p

Highly efficient antigen presentation to CD4⁺ and CD8⁺ T cells is limited to the early phase of infection following s.c. viral infection.

<p>2×10⁶ CFSE-labelled purified CD4⁺ and CD8⁺ T cells specific for the HA epitopes of influenza were adoptively transferred into naïve BALB/c mice infected on days 1 to 5 respectively with PR8 influenza virus. Proliferation of T cells in the popliteal lymph node was indicated by dilution of the CFSE stain 3 days after transfer. <i>A.</i> Data show representative flow cytometric profiles of T cell proliferation from popliteal lymph node from one of two similar experiments and <i>B,</i> Histogram shows the mean number of dividing T cells in popliteal lymph node±SEM 3 days after transfer. Data are pooled from two experiments showing 5 mice for HNT CD4⁺ expansion and 6 mice for CL4 CD8⁺ expansion.</p

CD8α DC dominate antigen presentation to naïve gBT-I CD8⁺ T cells following s.c. infection.

<p>C57BL/6 mice were infected with WSN-gB by s.c. injection into the footpad. 48 and 72 h after infection, the popliteal lymph node were isolated and non-DC lineages were removed by antibody depletion. <i>A,B.</i> CD11c⁺ DC subsets were purified by flow cytometric sorting into DN DC, CD8α DC and pDC before culturing titrating numbers of each subset with 5×10⁴ CFSE-labelled gBT-I CD8⁺ T cells. Proliferation was analysed at 60 h of culture. Data show the mean and SEM of three independent experiments. <i>C,D.</i> Lymph node-resident CD8α DC and not skin-derived trafficking DC, prime naïve CD8⁺ T cells following s.c. infection with WSN-gB. C57BL/6 mice were infected with 400 PFU by s.c. injection into the footpad. At 48 h (<i>C</i>) and 72 h (<i>D</i>) following infection, DC were enriched from popliteal lymph node and flow cytometrically sorted into Langerhans cells (CD205⁺CD8α^+/−), dermal DC (CD205^intCD8α⁻), CD8α DC (CD205⁺CD8α⁺) or DN DC (CD205⁻CD8α⁻). Titrating numbers of purified DC subsets were cocultured with 5×10⁴ CFSE-labelled gBT-I CD8⁺ T cells. The x-axis shows the range of DC used as stimulators over different experiments. Proliferation was analysed at 60 hr of culture. Data are pooled from three independent experiments and show the mean and SEM. Significant differences (<i>p</i>≤0.05) in T cell proliferation induced by different DC populations are indicated by an asterisk.</p

Dendritic cell subsets mediating MHC class I and class II-restricted antigen presentation differ following intravenous infection.

<p><i>A,B,</i> 24 h after infection with PR8 virus, non-DC were depleted from spleens and the remaining cells were purified by flow cytometry into CD8α DC (CD8α⁺CD45RA⁻), CD4 DC (CD4⁺CD45RA⁻), and pDC (CD8α⁻CD4⁻CD45RA⁻); or <i>C,D</i> alternately, CD8α DC (CD8α⁺CD4⁻), CD4 DC (CD8α⁻CD4⁺) or DN DC (CD8α⁻CD4⁻) subsets. Titrating numbers of each subset was cocultured with 5×10⁴ CFSE-labelled HA-specific CD8⁺ or CD4⁺ T cells and the amount of proliferation was measured at 60 h of culture by the loss of CFSE fluorescence. Data show the mean±SEM of three (A,C) to four (B,D) independent experiments. Significant differences (p≤0.05) in proliferation induced by different DC populations are indicated by an asterisk.</p

Mutations in nsP1 and PE2 are critical determinants of Ross River virus-induced musculoskeletal inflammatory disease in a mouse model

AbstractThe viral determinants of alphavirus-induced rheumatic disease have not been elucidated. We identified an RRV strain (DC5692) which, in contrast to the T48 strain, does not induce musculoskeletal inflammation in a mouse model of RRV disease. Substitution of the RRV T48 strain nonstructural protein 1 (nsP1) coding sequence with that from strain DC5692 generated a virus that was attenuated in vivo despite similar viral loads in tissues. In contrast, substitution of the T48 PE2 coding region with the PE2 coding region from DC5692 resulted in attenuation in vivo and reduced viral loads in tissues. In gain of virulence experiments, substitution of the DC5692 strain nsP1 and PE2 coding regions with those from the T48 strain was sufficient to restore full virulence to the DC5692 strain. These findings indicate that determinants in both nsP1 and PE2 have critical and distinct roles in the pathogenesis of RRV-induced musculoskeletal inflammatory disease in mice