Dissociation protocols influence the phenotypes of lymphocyte and myeloid cell populations isolated from the neonatal lymph node

Frequencies and phenotypes of immune cells differ between neonates and adults in association with age-specific immune responses. Lymph nodes (LN) are critical tissue sites to quantify and define these differences. Advances in flow cytometry have enabled more multifaceted measurements of complex immune responses. Tissue processing can affect the immune cells under investigation that influence key findings. To understand the impact on immune cells in the LN after processing for single-cell suspension, we compared three dissociation protocols: enzymatic digestion, mechanical dissociation with DNase I treatment, and mechanical dissociation with density gradient separation. We analyzed cell yields, viability, phenotypic and maturation markers of immune cells from the lung-draining LN of neonatal and adult mice two days after intranasal respiratory syncytial virus (RSV) infection. While viability was consistent across age groups, the protocols influenced the yield of subsets defined by important phenotypic and activation markers. Moreover, enzymatic digestion did not show higher overall yields of conventional dendritic cells and macrophages from the LN. Together, our findings show that the three dissociation protocols have similar impacts on the number and viability of cells isolated from the neonatal and adult LN. However, enzymatic digestion impacts the mean fluorescence intensity of key lineage and activation markers that may influence experimental findings

Neonatal CD8 T-cell Hierarchy Is Distinct from Adults and Is Influenced by Intrinsic T cell Properties in Respiratory Syncytial Virus Infected Mice

Following respiratory syncytial virus infection of adult CB6F1 hybrid mice, a predictable CD8+ T cell epitope hierarchy is established with a strongly dominant response to a Kd-restricted peptide (SYIGSINNI) from the M2 protein. The response to KdM282-90 is ∼5-fold higher than the response to a subdominant epitope from the M protein (NAITNAKII, DbM187-195). After infection of neonatal mice, a distinctly different epitope hierarchy emerges with codominant responses to KdM282-90 and DbM187-195. Adoptive transfer of naïve CD8+ T cells from adults into congenic neonates prior to infection indicates that intrinsic CD8+ T cell factors contribute to age-related differences in hierarchy. Epitope-specific precursor frequency differs between adults and neonates and influences, but does not predict the hierarchy following infection. Additionally, dominance of KdM282-90 –specific cells does not correlate with TdT activity. Epitope-specific Vβ repertoire usage is more restricted and functional avidity is lower in neonatal mice. The neonatal pattern of codominance changes after infection at 10 days of age, and rapidly shifts to the adult pattern of extreme KdM282- 90 -dominance. Thus, the functional properties of T cells are selectively modified by developmental factors in an epitope-specific and age-dependent manner

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

Quantitative and qualitative deficits in neonatal lung-migratory dendritic cells impact the generation of the CD8+ T cell response.

CD103+ and CD11b+ populations of CD11c+MHCIIhi murine dendritic cells (DCs) have been shown to carry antigens from the lung through the afferent lymphatics to mediastinal lymph nodes (MLN). We compared the responses of these two DC populations in neonatal and adult mice following intranasal infection with respiratory syncytial virus. The response in neonates was dominated by functionally-limited CD103+ DCs, while CD11b+ DCs were diminished in both number and function compared to adults. Infecting mice at intervals through the first three weeks of life revealed an evolution in DC phenotype and function during early life. Using TCR transgenic T cells with two different specificities to measure the ability of CD103+ DC to induce epitope-specific CD8+ T cell responses, we found that neonatal CD103+ DCs stimulate proliferation in a pattern distinct from adult CD103+ DCs. Blocking CD28-mediated costimulatory signals during adult infection demonstrated that signals from this costimulatory pathway influence the hierarchy of the CD8+ T cell response to RSV, suggesting that limited costimulation provided by neonatal CD103+ DCs is one mechanism whereby neonates generate a distinct CD8+ T cell response from that of adults

Modulating CD28-mediated costimulatory signals differentially affects K^dM2_82–90 and D^bM_187–195-specific responses.

<p>A) Mice were infected with RSV on day 0 and subsequently given IP injections of 20 µg each of antibodies against CD80 and CD86 at the indicated day post-infection. Epitope-specific CD8+ T cell responses were measured by surface and tetramer staining at 7 days post-infection. All groups were compared using a two-way ANOVA and Tukey's multiple comparisons tests, and there were no significant differences in the D^bM_187–195 response. Differences between the K^dM2_82–90 response on Day 2 and other groups are indicated below the figure (* p≤0.05, *** p≤0.001, **** p≤0.0001). B) The response ratio/immunodominance profile of CD8+ T cell responses was obtained for each mouse by dividing the K^dM2_82–90 response by the response to D^bM_187–195. * indicates p≤0.05 following one-way ANOVA and Dunnett's multiple comparisons test. C) Day 7 CD8+ T cell responses of mice injected with the indicated dose of anti-CD80 and CD86 at day 2 post-infection or isotype control antibodies (either 50 µg or 20 µg). All groups were compared using a two-way ANOVA and Tukey's multiple comparisons tests. Differences in the K^dM2_82–90 response between the 50 and 25 µg groups and all other groups are indicated below the figure (ns not significant, ** p≤0.01, and **** p≤0.0001). The D^bM_187–195 responses are significantly different between both the 50 and the 25 µg groups and isotype (p≤0.01) only. D) CD8+ T cell response ratios of mice treated with each dose of anti-CD80 and CD86 antibodies. Groups were compared with a one-way ANOVA and Tukey's multiple comparisons test (** p≤0.01, *** p≤0.001, **** p≤0.0001), and all error bars represent the SEM.</p

Uptake and processing of soluble antigen is age-dependent.

<p>Soluble ovalbumin-FITC protein (A and B) or ovalbumin-DQ (C and D) were co-administered at the time of RSV infection and the percent of the CD103+ and CD11b+ DC populations positive for FITC expression (A or B) or shown to be processing antigen by unquenching of ova-DQ were measured in the MLN at days 1, 2, and 3 post-infection. Data are combined from two independent experiments using pooled lymph node samples (3–7 mice/pool depending on age and availability) and error bars indicate the SEM. * p≤0.05, **p≤0.01, ***p≤0.001, **** p≤0.0001 by two-way ANOVA and Tukey's multiple comparisons test.</p

RSV infection of lung migratory dendritic cell populations in the lung and draining mediastinal lymph node.

<p>Percent of infected (GFP+) dendritic cell populations in the lung (A and B) and the posterior mediastinal lymph node (MLN, C and D) at days 1–3 post-infection. Lung and MLN DC populations were gated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003934#ppat.1003934.s001" target="_blank">Figure S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003934#ppat.1003934.s002" target="_blank">S2</a>, respectively. Percent GFP+ of CD103+ DCs (A and C) and CD11b+ DCs (B and D) was determined by comparison to mice infected at the same time with wtRSV. Data is representative of two experiments done with two pooled samples of 3–7 mice each and the error bars represent the SEM. * p≤0.05, **p≤0.01, ***p≤0.001, ns is no significance by two-way ANOVA and Sidak's multiple comparisons test.</p

Lung-migratory dendritic cell responses change dramatically during early life.

<p>CD103+ and CD11b+ DC populations in the MLN three days post-RSV infection were measured in mice of different ages. A) DC populations as a percent of CD11c+-gated cells of mice infected at the indicated age. B) Ratio of CD103+ to CD11b+ cells in the MHC Class II high gate. C) Total number of DC cells acquired on the flow cytometer per mouse after running samples to completion. Data in A and C are representative of three experiments using mice at available ages, and B represents data compiled from three independent experiments. Groups were compared using a two-way or one-way ANOVA and Tukey's multiple comparisons tests in GraphPad Prism and significant differences within the 7–21 day transition period are indicated (* p≤0.05, ** p≤0.01, ***p≤0.001, **** p≤0.0001). Error bars represent the SEM.</p

Neonatal CD103+ DCs differentially induce RSV-specific adaptive CD8+ T cell responses.

<p>A) CFSE-labeled TCR transgenic CD8+ T cells specific for K^dM2_82–90 or D^bM_187–195 were co-cultured in a 10∶1 ratio with CD103+ dendritic cells sorted from the lymph nodes of RSV infected adults or neonates one day after infection. The percent of cells that proliferated following three days in co-culture was calculated using the proliferation module in FlowJo 9.4.10. Negative control samples consisted of TCR Tg T cells co-cultured with splenocytes with no exogenous peptide, and positive control samples were co-cultured with splenocytes and 10⁻⁶M of specific peptide. B) Comparison of the ratio of proliferation (percent of K^dM2_82–90-specific cells that proliferated/percent of D^bM_187–195-specific cells that proliferated) induced by flow-sorted neonatal vs. adult CD103+DCs. Groups were compared using a student's t-test.</p

Age-dependent phenotypic changes in the CD103+ and CD11b+ DC populations in the MLN three days post-infection.

<p>A) Evaluation of the Class II high populations of neonatal (7 days old) vs. adult mice shows differences in composition and phenotype of DC populations. B) Costimulatory molecule (CD86, CD80 and CD70) expression was measured by flow cytometry on CD103+ and CD11b+ DCs three days post-infection at the indicated age and the mean fluorescence intensity (MFI) is displayed. Data are representative of three experiments using two to three samples of lymph nodes pooled from 3–7 mice. Error bars indicate the SEM, and all data was analyzed using one-way ANOVA with Tukey's multiple comparisons test (* p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001).</p