Our results have demonstrated that ∼50% of infected cells establish latency immediately upon integration of provirus, a phenomenon termed early latency, which appears to occur by mechanisms that are distinct from epigenetic silencing observed with HIV provirus that establishes productive infections. In this study, we have used a mini-dual HIV reporter virus (mdHIV) to compare the long-term stability of provirus produced as early latent or productive infections using Jurkat-Tat T cell clones. Cloned lines bearing mdHIV provirus integrated at different chromosomal locations display unique differences in responsiveness to signaling agonists and chromatin-modifying compounds, and they also produce characteristic expression patterns from the 5′ long terminal repeat (LTR) dsRed and internal EIF1α-enhanced green fluorescent protein (EIF1α-eGFP) reporters. Furthermore, reporter expression profiles of single cell sorted subcultures faithfully reproduce expression profiles identical to that of their original parental population, following prolonged growth in culture, without shifting toward expression patterns resembling that of cell subclones at the time of sorting. Comparison of population dispersion coefficient (CV) and mean fluorescence intensity (MFI) of the subcloned lines showed that both untreated and phorbol myristate acetate (PMA)-ionomycin-stimulated cultures produce expression patterns identical to those of their parental lines. These results indicate that HIV provirus expression characteristics are strongly influenced by the epigenetic landscape at the site of chromosomal integration.Understanding the mechanisms of HIV proviral latency is essential for development of a means to eradicate infection and achieve a cure. We have previously described an IMPORTANCE There is currently considerable interest in development of therapies to eliminate latently infected cells from HIV-infected patients on antiretroviral therapy. One proposed strategy, known as “shock and kill,” would involve treatment with therapies capable of inducing expression of latent provirus, with the expectation that the latently infected cells could be killed by a host immune response or virus-induced apoptosis. In clinical trials, histone deacetylase (HDAC) inhibitors were shown to cause reactivation of latent provirus but did not produce a significant effect toward eliminating the latently infected population. Results shown here indicate that integration of HIV provirus at different chromosomal locations produces significant effects on the responsiveness of virus expression to T cell signaling agonists and chromatin-modifying compounds. Given the variety of phenotypes produced by integrated provirus, it is unlikely that any single potential shock-and-kill therapy will be effective toward purging the latently infected population. Human immunodeficiency virus (HIV) infection is presently incurable because the virus establishes latent infections of resting memory immune cells in which provirus expression has become transcriptionally silenced . Despite+ T cells also express several microRNAs (miRNAs), which target the 3′ end of HIV-1 transcripts that can contribute to transcriptional silencing of the provirus  alone or in combination with ionomycin  for 24 h where indicated below. HEK293T cells were cultured in Dulbecco modified Eagle medium  with 10% fetal bovine serum supplemented with penicillin (100 U/ml), streptomycin (100 U/ml), and amphotericin B . The mini-dual-fluorescence HIV (mdHIV) reporter virus expresses dsRed from the 5′ HIV LTR and enhanced GFP (eGFP) from an internal EIF1α promoter to enable detection of infected cells independently from LTR expression  cells were obtained from the American Type Culture Collection. Jurkat cells were grown in RPMI 1640 (Sigma) supplemented with 10% fetal bovine serum (Sigma), penicillin (100 U/ml), streptomycin (100 μg/ml), and amphotericin B  (Gibco). Jurkat-Tat cells were grown in the same with an additional supplement of Geneticin . Cell cultures were maintained in a humidified 37°C incubator with a 5% COpression  23). Th. Th2 atmpression . Vesiculpression , 23. Vir6 Jurkat-Tat cells were infected at a multiplicity of infection (MOI) of ∼0.1 for 1 h. Infected Jurkat-Tat cells were monitored for eGFP and dsRed expression every day for 4 days and once weekly thereafter for a month, using a BD Biosciences LSR II system as previously described .Integration sites of mdHIV latently infected Jurkat-Tat clones were determined by a restriction enzyme PCR strategy using the scheme shown in Fig. S4 in the supplemental material and as previously described . Brieflynd WB215 . Amplicond WB215 , digeste+) plasmid construct was described previously  β-galactosidase  internal control plasmid. The cells were incubated for 48 h and exposed to PMA for 24 h prior to lysis of the cells and assay for luciferase and β-galactosidase activity , which included both latently and productively infected cells, was resorted for single cells into 96-well plates, and the clones were expanded over the following 2 months , in which dsRed is expressed from the 5′ LTR and eGFP from an internal EIF1α promoter to enable detection of infected cells independently from LTR expression  23). We. We23). tutively . With th2 months , 23.From these efforts, we selected 16 parental lines that produced a broad variety of PMA-induced expression of dsRed for further analysis, for which we calculated a mean fluorescence intensity (MFI) of LTR-dsRed expression produced in both untreated cells and cells treated with PMA for 24 h. As shown in We also compared responsiveness of the 16 parental cell lines to the effect of tumor necrosis factor alpha (TNF-α) and found that the response roughly correlated for the two agonists in many of the lines but not for lines 131, 49, 90, 61, and 53, which produced a proportionately lower response to TNF-α than to PMA . In geneWe confirmed that with the exception of two clones (60 and 25), the parental cell lines that produce these distinctive phenotypes represent unique single integration sites of proviral genomes, by determining the sequence of chromosomal DNA flanking the LTR for 12 of the lines . Consistent with previous studies , 34, we + dsRed−) or productively infected (eGFP+ dsRed+) cells, in addition to cells in which both reporters had become repressed or silenced (eGFP− dsRed−) . Although we obtained subclones from resorting each of the parental lines, we recovered subclones representing all three expression patterns only for parental lines 77 and 90  for single cells that represented latently infected  and dispersion coefficient of variation (CV) for both reporters, at 6 weeks after isolation of the subclones produced from the productively and latently infected or silenced populations, and compared these to values for the parental line . By this+/dsRed−), productive (eGFP+/dsRed+), and silenced (eGFP−/dsRed−) populations for each of the cell lines, where available (+ dsRed−) and , particularly for the untreated cultures  in parallel with further analysis of line 77. For this experiment, we expanded a single clone, from the latent  reporter and measured expression of luciferase in untreated and PMA-stimulated cells. Transfection efficiency was normalized by cotransfection of an RSV β-galactosidase internal control reporter gene plasmid. This plasmid is responsive to Tat function, as we observed significantly higher luciferase expression in Jurkat-Tat cells than in Jurkat cells (r (G418 resistance) marker   which doat cells . In tranat cells . We do n) marker . Consequ+ for several hours until this signal decays, after which the GFP signal is dependent upon expression from the LTR (+ GFP−) (+ GFP+) for several weeks postinfection. However, the proportion of total infected cells in both cultures decreased sharply 2 weeks postinfection, which likely represented cytopathic effects of viral gene products. Nine days postinfection, cells harboring productive infections (mCherry+ GFP+) were eliminated, or virus expression had reverted to latency in both cultures. Additionally, the proportion of latently infected cells (mCherry+ GFP−) stabilized at a constant level by day 14 and persisted for at least two further weeks. These results indicate that HIV is able to establish early latency immediately upon infection and maintain a stable population of these cells, even in cells that constitutively express Tat. This implies that mechanisms required for production of this mode of latency may be different than those previously described for HIV that productively replicates upon infection  , or thosnfection –40.There is currently a consensus that a cure for HIV disease will require a means of purging, or effectively eliminating, latently infected cells, which are unaffected by current therapies and can act as a reservoir for viral rebound in HIV-infected individuals . One strIn this study, we have examined the possibility that the site of chromosomal integration can affect the phenotypic expression of HIV provirus, by isolating multiple clonal cell lines harboring a single-copy integrant of a mini-dual-fluorescence reporter HIV (mdHIV) . Cloned Our results have important implications for the proposed shock-and-kill strategy to eliminate latently infected cells from patients. First, it appears that provirus integrated at different chromosomal locations will require different shock treatments to produce reactivation. For example, provirus integrated at some locations may be reactivated by HDAC inhibitors alone, while others may require demethylation at histone H3K9 and/or H3K27 , 45. StiZNF721 gene, although in the reverse orientation , productively replicating (dsRed+ eGFP+), or silenced provirus (dsRed− eGFP−), recapitulated the identical full basal expression profile of the parental line within several weeks of subculture. We have observed this effect with at least five of the parental lines, each of which produces a unique basal HIV expression phenotype, and this indicates that transcription factors that bind the LTR in unstimulated cells must restore transcription levels to the original basal state, despite any potential influences from flanking regulatory regions at the site of HIV integration. Thus, while the site of integration can influence responsiveness to various signaling and chromatin-modifying agonists, HIV LTR function seems to dominate over the flanking environment for basal expression. It is somewhat surprising that cells isolated from the productively infected population (dsRed+ eGFP+) gradually revert to the parental basal phenotype, given that these cells constitutively express Tat. This finding implies that there must be mechanisms that can overcome the effect of Tat function to enable LTR silencing and reestablishment of latency. Such mechanisms might be similar, if not identical, to those involved in establishment of early latency in newly infected cells, a feature we discuss in more detail below. We found that in general, cells cloned from the productively infected (dsRed+ eGFP+) and latently infected (dsRed− eGFP+) cultures revert back to the parental phenotype more rapidly than do cells from clones of the HIV-silenced population (dsRed− eGFP−). A possible explanation for this finding is that the silenced provirus population, which has lost expression of both the LTR and internal EIF1α promoters, may have accumulated epigenetic marks that take longer to reverse in culture than the latently and productively infected populations. Taken together, these findings suggest that there is a strong propensity for HIV provirus to maintain a latent state in infected but unstimulated cells.Most of the cloned parental lines generate significantly diverse basal expression for both the LTR and internal EIF1α promoters, likely representing transcriptional noise generated by a stochastic signaling environment in unstimulated cells , 51. We Several studies have demonstrated that the positive-feedback effect of Tat on viral transcription is critical for overcoming mechanisms that promote latency of HIV-1 provirus , 38, 52,A better understanding of the mechanisms that contribute to establishment and maintenance of the latent HIV reservoir is necessary for development of therapies to eradicate the latently infected population from patients with HIV disease. In this study, we have directly examined the role of HIV integration sites in expression phenotypes of provirus and found significant differences in responsiveness to T cell signaling agonists and chromatin-modifying compounds. Our results are consistent with the view that the latent HIV-infected cell population is not only very heterogeneous but also stable and that a combination of therapies will be necessary to eradicate latent HIV-infected cells from patients with HIV/AIDS disease.


Oncorhynchus nerka) stocks throughout the southern part of their North American range have experienced declines in productivity over the past two decades. In this study, we tested the hypothesis that pink (O. gorbuscha) and chum (O. keta) salmon stocks have also experienced recent declines in productivity by investigating temporal and spatial trends in productivity of 99 wild North American pink and chum salmon stocks. We used a combination of population dynamics and time series models to quantify individual stock trends as well as common temporal trends in pink and chum salmon productivity across local, regional, and continental spatial scales. Our results indicated widespread declines in productivity of wild chum salmon stocks throughout Washington (WA) and British Columbia (BC) with 81% of stocks showing recent declines in productivity, although the exact form of the trends varied among regions. For pink salmon, the majority of stocks in WA and BC (65%) did not have strong temporal trends in productivity; however, all stocks that did have trends in productivity showed declining productivity since at least brood year 1996. We found weaker evidence of widespread declines in productivity for Alaska pink and chum salmon, with some regions and stocks showing declines in productivity  and others showing increases . We also found strong positive covariation between stock productivity series at the regional spatial scale for both pink and chum salmon, along with evidence that this regional-scale positive covariation has become stronger since the early 1990s in WA and BC. In general, our results suggest that common processes operating at the regional or multi-regional spatial scales drive productivity of pink and chum salmon stocks in western North America and that the effects of these process on productivity may change over time.Sockeye salmon ( Gadus morhua)[Oncorhynchus spp.)[Clupea pallasi)[O. nerka) after the 1976/77 climatic regime shift [The influence of environmental change on fish population productivity is a central problem in fisheries science . Long tis morhua),4, Pacifhus spp.),6, Pacif pallasi),8, and s pallasi),10. Someme shift ,11. In ome shift , potentime shift .Detecting trends in fish stock productivity in a timely manner is difficult because short-term variability in recruitment and noisy abundance data usually combine to obscure changes in mean productivity of individual stocks ,15. AlthO. gorbuscha), and chum salmon (O. keta) tend to be influenced similarly by changes in ocean conditions , suggesting that productivity of pink and chum salmon stocks in WA and British Columbia (BC) may have also declined over the past two decades [For sockeye salmon, multi-stock analyses have indicated widespread declines in productivity over the past two decades for stocks ranging from Washington (WA) to Southeast Alaska . These o decades . However decades , possibl decades .In this study, we asked whether productivity of wild North American pink and chum salmon stocks has declined over the past two decades and if so what is the spatial extent of the declines. In particular, our objectives were (1) quantify temporal trends in productivity for pink and chum salmon stocks throughout their North American range to determine the magnitude and direction of recent temporal shifts, and (2) investigate the extent of spatial heterogeneity in productivity trends to better understand the mechanisms driving temporal trends. To accomplish these objectives, we first estimated stock productivity trends for each of 99 pink and chum salmon stocks using a modified stock-recruit model and Kalman filter fitting procedure. We then estimated common productivity trends across all stocks using dynamic factor analysis (DFA). Together, this two-step modeling approach allowed us to quantify and summarize temporal trends in pink and chum salmon productivity across local, regional, and continental scales.We used wild spawner abundance and total recruitment (catch plus escapement) data for 53 chum salmon stocks  and 46 pOut of the 53 chum salmon data sets, 36 did not have full age-structured data. Without age-structure information, total returns in a given year cannot be properly allocated to a particular brood year of spawners. Therefore, to estimate total recruitment, we used assumptions about the age-structure of returning adults. Following Pyper et al. , we usedWe chose to assume all returns were age-4 when little age data were available based on four lines of evidence that suggested this was an appropriate assumption. First, the dominant age of returning adults across the 17 stocks with full age data was age-4 , with most chum salmon returning as age-3 and age-4 for WA and BC stocks  and most salmon returning as age-4 and age-5 for Alaska stocks . Second, recruitment series reconstructed using an age-4 assumption are strongly and positively correlated with observed recruitment series. For example, the average correlation between recruitment estimates calculated from observed age data and estimates reconstructed assuming all returns were age-4 across each of the 17 stocks with known age data was 0.82 . Third, α parameter values estimated using a Kalman filter procedure.We used two methods to estimate stock-specific time series of productivity for the 99 pink and chum salmon stocks. The first set of productivity indices were the residuals from a stationary Ricker spawner-recruit model, whereas the second set were time-varying Ricker St is the number of spawners in brood year t, Rt is the number of recruits resulting from St, α is (constant) productivity at low spawner densities, β is the density-dependent effect, and et is the residual error assumed to be normally distributed with 0 mean and variance We used residuals from a stationary Ricker spawner-recruit model as estimates of annual stock productivity, which removed potential within-stock density-dependent effects ,25,26. Fα parameter values for each stock using a Kalman filter procedure [α parameter) [α parameter, i.e.,Rt is the number of recruits in brood year t, St is the number of spawners, αt is the annual stock productivity at low spawner abundances, β is the density dependence coefficient, and vt is the observation error assumed to be normally distributed with a mean 0 and variance αt parameter,wt is the process error assumed to be normally distributed with mean 0 and variance We estimated time-varying Ricker rocedure ,28. The rocedure . The starameter) . The obsl trends .αt parameter using an iterative Bayesian updating procedure whereby the prior value for αt  was updated based on how well at−1 predicted the following year’s loge(Rt/St) [α parameter where each αt value only depended on data up through year t. Following Peterman et al. [αt parameters. Unlike the Kalman filter procedure, which estimated the earliest αt value first and moved forward through time, the smoothing procedure worked backwards starting with the most recent filtered αt value [αt value, the smoother calculated a weighted average between the estimate of the filtered value at time t and the smoothed estimate at time t + 1, thereby producing estimates of αt that were based on the whole time series, rather than only the previously estimated values. This smoothed time series of αt, standardized to a mean of 0 and a standard deviation of 1, was used as our second stock-specific productivity index.The Kalman filter model estimated annual values for the e(Rt/St) . This prn et al. , we thenαt value . For eacThe other parameters of the Kalman filter model,  given a set of multivariate time series, which unlike other time series analyses , can be used to analyze short, non-stationary time series with missing values [yt is the vector of observed data at time t, τt is an M x 1 vector of common trends, Z is the stock-specific factor loadings for each trend, which describe the form of the linear combination of trends, et is the observation error term assumed to be normally distributed with mean 0 and variance-covariance matrix R, and ft is the process error also assumed to be normally distributed with mean 0 and variance-covariance matrix Q. In our study, all DFA trends were modeled using a random walk and were estimated using a Kalman filter/smoothing algorithm, which restricted the trends to smooth curves [To more clearly identify shared trends in productivity, we used DFA models, which are a class of multivariate autoregressive state-space models, to estimate common productivity trends across all stocks within a species. Dynamic factor analysis aims to estimate a specific number of common trends  including (1) a diagonal and equal variance-covariance matrix where all stock variances were equal and covariances between stocks were assumed 0, (2) a diagonal and unequal matrix where the variances were allowed to vary between stocks and covariances were assumed 0, (3) an equal matrix where the variances were all equal and the covariances were allowed to be non-zero but were all equal, and (4) a north-south matrix, which was similar to the equal variance-covariance matrix but variances and covariances differed for northern stocks (AK) and southern stocks (BC and WA) separately.We fit a total of 24 DFA models for each species using different combinations of trends and observation error variance-covariance matrices. All DFA models were fit using the residuals from the stationary Ricker models, rather than the Kalman filter productivity indices, because the DFA model already includes a Kalman filtering procedure. All productivity series were truncated to the period after 1979 because many stocks had considerable missing data or no data prior to 1980 . We fit models with 1–6 common trends and four different observation error variance-covariance matrices  as a model selection criterion [C values were evaluated based on four criteria: (1) the most parsimonious model was defined as the model with the lowest AICC value, (2) equally plausible models were defined as models with AICC values within 3 AICC units of the lowest, (3) less plausible models had AICC values greater than 3 from the minimum and were rejected with caution, and (4) models with AICC values greater than 10 compared to the minimum were rejected with confidence [We used the small-sample Akaike Information Criterion  compared to chum salmon  positive covariation between stocks within a region, (2) positive covariation between stocks in different regions in WA and BC, although this pattern was not as strong for chum salmon αt series, and (3) weak covariation between stocks that enter the ocean at distant locations  productivity for the majority of chum salmon stocks in WA and BC has declined over the past two decades, although the functional form of declines varied across regions with Central BC stocks showing an abrupt and steep decline in productivity starting around brood year 2000 and stocks in the Outside WA and Northern BC regions showing a more gradual declining trend, (2) trends in productivity for AK pink and chum salmon stocks were more variable with some regions and stocks showing declines in productivity and others showing increases, (3) there was strong positive covariation of productivity series within regions, and (4) covariation between productivity series for stocks in WA and BC has become stronger in recent years for both pink and chum salmon. In general, our results suggest that productivity of pink and chum salmon stocks is driven by common processes operating at the regional or multi-regional spatial scale, and the effects of these drivers may not be constant through time.Evidence for widespread declines in productivity was stronger for chum salmon than for pink salmon. With the exception of the Kalman filter results for Inside WA chum salmon stocks, both the DFA and Kalman filter analyses indicated that chum salmon stocks throughout WA and BC have experienced declines in productivity over the past two decades. In contrast, the Kalman filter analysis for pink salmon indicated that the low frequency signal  tended to contribute less to the total variability for many stocks in WA and BC. This corresponded with the single common DFA trend for pink salmon that did not show strong increasing or decreasing trends in productivity over the past two decades. However, across the WA and BC pink salmon stocks where a trend in productivity was observed, the trend was consistently downward over the past two decades. This finding that most chum and some pink salmon stock productivities have declined markedly throughout WA and BC is consistent with the findings of Peterman and Dorner  that indαt series indicated above average or increasing productivity trends for all three Southeast Alaska pink salmon stocks since at least brood year 1990 . One potential explanation for why productivity trends of southern Puget Sound chum salmon stocks are different is differences in physical and biological conditions in different parts of Puget Sound. For example, Duffy et al. [We observed a general increasing trend in productivity for chum salmon stocks in the Inside WA region, which was opposite of the declining productivity trends observed for chum salmon stocks in the four other geographic regions in WA and BC. More specifically, the South Sound and Hood Canal chum salmon stocks had increasing trends in the Kalman filter y et al.  indicatey et al.  indicatey et al. , improvey et al. , and chay et al. ,50. ThesSimilar to Peterman and Dorner  and DornThe observed patterns of covariation in productivity are also consistent with previously reported covariation patterns for wild pink and chum salmon abundances ,58. In pOur results broadly agree with those of several previous studies that suggested spatial covariation between demographic rates of nearby sockeye and chinook salmon stocks have increased in recent decades ,39,59. Iα parameter often leads to overestimates of the exploitation rate for a stock compared to using a Kalman filter fitting procedure [α parameter put more weight on historical data compared to the Kalman filter model, which tends to update parameter estimates faster [Regardless of the underlying mechanisms driving the trends, our results indicate that productivity of most chum salmon and some pink salmon stocks is non-stationary, which has important management and conservation implications if non-stationarity is not accounted for when setting management targets. Our results support the idea that the use of stationary spawner-recruit models to estimate stock productivity may be inappropriate for setting management targets, such as harvest rates or escapement goals, particularly in situations where large changes in productivity have occurred, such as those observed for chum salmon throughout BC ,31. Inderocedure . This ovs faster ,31.In conclusion, our results indicate that the majority of North American chum and some pink salmon stocks have experienced large shifts in productivity over the past two decades, and at least for WA and BC stocks, these shifts have been downward. The coherence of these declines over regional and multi-regional spatial scales suggests that physical or biological factors operating at similar spatial scales are likely driving the observed trends. Because productivity is an important determinant of marine and anadromous fish stock dynamics, the sharp or gradual shifts in productivity observed here for pink and chum salmon can have important consequences for management and conservation. In particular, this research further demonstrates the need to account for time varying demographic rates in stock assessments to quickly detect temporal changes that may otherwise lead to an increased risk of overexploitation.S1 Figr) and Recruitment series are standardized to a mean of 0 and standard deviation of 1. Correlation coefficients between the two recruitment series are given within each panel ((PDF)Click here for additional data file.S2 FigThe residual series are standardized to a mean of 0 and standard deviation of 1. The ordinate gives the stock, which are arranged south (bottom) to north (top) and grouped by geographic region. The area of the circle indicates the magnitude of the productivity values. Open circles represent negative values (red) and filled circles indicate positive values (blue).(PDF)Click here for additional data file.S3 FigThe residual series are standardized to a mean of 0 and standard deviation of 1. The ordinate gives the stock, which are arranged south (bottom) to north (top) and grouped by geographic region. The area of the circle indicates the magnitude of the productivity values. Open circles represent negative values (red) and filled circles indicate positive values (blue).(PDF)Click here for additional data file.S4 FigCorrelations were calculated using all available brood years. The magnitude of the correlation is given by the area of the circle with larger circles representing larger correlations than smaller circles. Negative correlations between stocks are shown as open circles (red) and positive correlations are shown as filled circles (blue). Stocks are grouped by geographic region, which are arranged south  to north .(PDF)Click here for additional data file.S5 FigCorrelations were calculated using all available brood years. The magnitude of the correlation is given by the area of the circle with larger circles representing larger correlations than smaller circles. Negative correlations between stocks are shown as open circles (red) and positive correlations are shown as filled circles (blue). Stocks are grouped by geographic region, which are arranged south  to north .(PDF)Click here for additional data file.S6 Figαt estimates from the Kalman filter model where each series is standardized to a mean of 0 and standard deviation of 1. Even and odd year brood lines are shown concurrently for each stock with the even year brood lines offset slightly above the odd year brood lines. The ordinate gives the stock, which are arranged south (bottom) to north (top) and grouped by geographic region. The area of the circle indicates the magnitude of the productivity values. Open circles represent negative values (red) and filled circles indicate positive values (blue). Small solid grey dots indicate zero values, whereas missing values are not shown.Each time series shows the smoothed (PDF)Click here for additional data file.S7 Fig(a) Even brood year (solid line) and odd brood year (dashed line) common productivity trends and (b) stock specific loadings for the even year  and odd year  common trends. In the bottom panel, stocks are ordered south (bottom) to north (top) and are grouped by geographic region.(PDF)Click here for additional data file.S8 Fig(a) between stock correlation coefficients for odd year runs and (b) correlations for even year runs. The magnitude of the correlation is given by the area of the circle with larger circles representing larger correlations than smaller circles. Negative correlations between stocks are shown as open circles (red) and positive correlations are shown as filled circles (blue). Stocks are grouped by geographic region, which are arranged south  to north .(PDF)Click here for additional data file.S1 Tableα and β give the parameter estimates from the best fit stationary Ricker model; Kalman filter αt gives the average αt value and SD gives the standard deviation for the αt series; Kalman filter S/N gives the signal-to-noise ratio for that stock.Brood years gives the range of years available for each stock; N gives the total brood years with data; R/S gives the average spawner to recruit ratio over all available brood years; Stationary (PDF)Click here for additional data file.S2 Tableα and β give the parameter estimates from the best fit stationary Ricker model; Kalman filter αt gives the average αt value and SD gives the standard deviation for the αt series; Kalman filter S/N gives the signal-to-ratio for that stock.Brood years gives the range of years available for each stock; N gives the total brood years with data; R/S gives the average spawner to recruit ratio over all available brood years; Stationary (PDF)Click here for additional data file.


TRBV/TRBJ gene segment usage varied markedly between individuals but remained relatively constant within individuals across the course of therapy. Despite this stable TRBV/TRBJ gene segment usage, a number of TCRβ sequence clonotypes showed dramatic changes in read frequency. These changes could not be linked to therapy outcomes in the present study; however, the TCRβ CDR3 sequences with the largest fold changes did include sequences with identical TRBV/TRBJ gene segment usage and high junction region homology to previously published CDR3 sequences from HCV-specific T cells targeting the HLA-B*0801-restricted 1395HSKKKCDEL1403 and HLA-A*0101-restricted 1435ATDALMTGY1443 epitopes. The pipeline developed in this proof of concept study provides a platform for the design of future experiments to accurately address the question of whether T cell responses contribute to SVR upon antiviral therapy. This pipeline represents a novel technique to analyze T cell dynamics in situations where conventional antigen-dependent methods are limited due to suppression of T cell functions and highly diverse antigenic sequences.To maintain a persistent infection viruses such as hepatitis C virus (HCV) employ a range of mechanisms that subvert protective T cell responses. The suppression of antigen-specific T cell responses by HCV hinders efforts to profile T cell responses during chronic infection and antiviral therapy. Conventional methods of detecting antigen-specific T cells utilize either antigen stimulation  or antigen-loaded tetramer staining. This limits the ability to profile T cell responses during chronic infection due to suppressed effector function and the requirement for prior knowledge of antigenic viral peptide sequences. Recently, high-throughput sequencing (HTS) technologies have been developed for the analysis of T cell repertoires. In the present study, we have assessed the feasibility of HTS of the TCRβ complementarity determining region (CDR)3 to track T cell expansions in an antigen-independent manner. Using sequential blood samples from HCV-infected individuals undergoing antiviral therapy, we were able to measure the population frequencies of >35,000 TCRβ sequence clonotypes in each individual over the course of 12 weeks.  The importance of T cell populations as mediators of protective immunity is well documented for a range of viral infections –3. To maDefining the role of HCV-specific T cells during both chronic infection and antiviral therapy is challenging due to limitations inherent in the experimental assays used to identify HCV-specific T cells. Conventional methods of detecting antigen-specific T cells utilize two basic techniques: (1) antigen stimulation  and (2) antigen-loaded tetramer staining. Antigen stimulation assays rely on a functional readout of T cell activity, however, chronic HCV infection and exogenous IFNα used during antiviral therapy are both associated with suppressed T cell function. This causes difficulty when assessing T cell responses in these groups of patients. Both antigen stimulation and tetramer staining also rely on prior knowledge of the specific antigenic peptides against which immune responses are directed. For a hugely diverse pathogen such as HCV, this means that even within a single-infected individual, it is unfeasible to profile immune responses targeting all antigenic sequence variants.eff) contracts and a proportion of these cells develop into memory T cell populations, which can persist for a number of years and can rapidly respond upon subsequent re-challenge with the same antigen . This recognition results in T cell activation, which together with co-stimulation signals, leads to the clonal expansion of viral-specific T cells that express cytokines and cytotoxic mediators to facilitate pathogen clearance. Following clearance of the invading pathogen, this clonal population of effector T cells  technologies have been developed for the analysis of CDR3 recombination events, enabling analysis of T cell populations with unprecedented detail. In the present study, we have assessed the feasibility of HTS of the TCRβ CDR3 region to track temporal changes in T cell populations in HCV-infected individuals undergoing antiviral therapy, a setting where conventional T cell assays are limited due to antigenic variation and T cell immune suppression. We describe a pipeline to identify expanding and contracting T cell clones from sequential blood samples and highlight the potential and pitfalls of HTS-based techniques to study T cell responses during infection.Clinical samples were used with informed consent, conforming to the ethical guidelines of the 1975 Declaration of Helsinki and study protocols were approved by the West of Scotland Research Ethics Committee. Blood samples were obtained from chronically HCV-infected patients who were beginning antiviral therapy as part of a larger clinical cohort . Blood s® VILO™ cDNA Synthesis Kit . RNA quality was assessed on an Agilent 2200 TapeStation , prior to reverse transcription. To amplify the TCR CDR3 region, a previously published set of primers was utilized consisting of 45 forward primers, each specific to a functional T cell receptor beta variable (TRBV) gene segment, and 13 reverse primers, each specific to a T cell receptor beta joining (TRBJ) gene segment , including 10% Q-solution, and pooled prior to library preparation.DNA-free total RNA was extracted from peripheral blood mononuclear cells (PBMCs) and reverse transcribed using the SuperScript segment . Duplica® Ampure® XP beads  and subsequently quantified on a Qubit® 2.0 Fluorometer using the Qubit® dsDNA High Sensitivity kit (Invitrogen). Library preparation, including end repair, A-tailing, and adaptor ligation, was performed using the KAPA HiFi Real-Time PCR Library Amplification kit for Illumina libraries . The A-tailed PCR amplicons were ligated to NEBNext® Multiplex Oligos for Illumina®  and end repair of the forked adaptors was carried out using either Index Primer Set 1 or 2 (New England Biolabs). Adaptor ligated products were quantified using the KAPA SYBR® FAST ABI Prism qPCR library quantification kit (KAPA Biosystems). Sequencing was performed using an Illumina MiSeq™ System Desktop Sequencer , with a 300 cycle MiSeq version 2 reagent kit (Illumina). To avoid errors due to low sequence diversity within the PCR amplicons the libraries were sequenced with a 25% PhiX spike-in, at a low cluster density (~800 K/mm2), and run metrics were checked to ensure that 85–90% of clusters passed quality filtering.PCR amplicons were washed, purified, and size-selected using AgencourtTRBV and TRBJ primer sequences were analyzed using custom perl scripts (SeqRenamer.pl and PrimerTrim.pl). The PrimerTrim.pl script trims the primer sequences from the reads, identifies reads lacking primer sequences, and outputs TCR Vβ and Jβ usage. Sequences lacking a TRBV or TRBJ primer pair or containing two TRBV or TRBJ primer sequences were removed using custom perl scripts (RemoveSamePrimer.pl and FastqFilter.pl), and a gene usage text file was generated from the PrimerTrim.pl output (using ParseStats.pl). The .fastq sequencing files were converted to .fasta files and identical reads were grouped into TCRβ sequence clonotypes using cd-hit-est (eff and memory T cell populations as opposed to low frequency naive T cell populations. Output files detailing productive sequence frequency for each sample were generated using a perl script (ParseIMGT.pl) and final data normalization and visualization was performed in R. All perl scripts used are publically available at https://github.com/josephhughes/TCRclust.Raw sequencing reads were trimmed, to remove poor quality sequence and adaptor sequences, using fastq-mcf-hit-est . For the-hit-est  to identThe results of the TCR sequencing and data processing are summarized in Table TRBV and TRBJ gene segment usage within sequence datasets, from each individual patient and time point, was used to calculate a distance matrix, using Manhattan distance measures, to provide a metric for the similarity in gene segment usage between datasets, as visualized for TRBV in Figure TRBJ in Figure TRBV and TRBJ gene segment usage was most similar  between samples collected at different time points from the same individual. In contrast, the TRBV and TRBJ gene segment usage was less similar  between samples from different individuals. The average distance value between intra-individual datasets from different time points for TRBV gene segment usage was 0.093 versus 0.221 for the inter-individual datasets . The average distance value between intra-individual datasets from different time points for TRBJ gene segment usage was 0.059 versus 0.146 for the inter-individual datasets . In all six patients the stability of the TCR repertoire, both in terms of numbers of TCRβ sequence clonotypes and of TRBV/TRBJ gene segment usage, is maintained despite a significant decrease in circulating lymphocytes across the course of therapy (data not shown), as previously identified during IFNα-based treatments  who only showed a reduction in viral load upon initiation of protease inhibitor treatment at week 4, a trend toward increased TCRβ sequence clonotype size was noted  or the nature of the infectious pathogen results in high inter- and intrahost antigenic variation . It is also valuable in settings (such as HCV treatment) where T cell numbers and function are suppressed. In the present study, we have developed a pipeline for the integration of sequential TCR HTS data, allowing analysis of multiple sequential datasets each containing over half a million sequence reads.TRBV and TRBJ gene usage over the course of HCV antiviral therapy, and the overall stability of the TCRβ repertoire within individuals. The clonotype-specific nature of this T cell response may contribute to the conflicting data regarding the importance of HCV-specific T cell responses during HCV therapy  infection , 35. FutTo avoid sampling or sequencing biases that may have hindered the identification of T cell expansion or contraction, we included the use of pooled PCR reactions and a high read frequency cut-off for the analysis of TCRβ sequence clonotypes. The use of unique molecular identifiers has been recently introduced to limit the effects of PCR bias in HTS experiments and this technique offers significant advantages to the analysis of future HTS studies of the TCR locus , 37. SamIn summary, the analysis of T cell responses through HTS techniques has the potential to greatly expand our understanding of the role of T cells in health and disease. The development of sequential analysis of TCRβ sequence clonotypes can provide an antigen-independent view of T cell dynamics in complex tissue environments, complementing existing T cell phenotyping assays, and providing insights into the role of immunological diversity in protection from infectious disease.MR contributed to study concept and design, acquisition and analysis of data, and wrote the manuscript. JH developed the bioinformatics pipeline and contributed to data analysis and manuscript writing. GW contributed technical and experimental support. RS, SB, and PM contributed to patient recruitment and administrative support. AP contributed to study supervision and funding. ET and JM contributed to study design, supervision, funding, and manuscript review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder involving aberrant proliferation of multiple tissues of neural crest origin. Retinal vascular alterations in NF1 have rarely been reported in the literature and their nature is not clear. This study describes distinctive retinal microvascular alterations and their relationship to choroidal nodules in patients with neurofibromatosis type 1.This was a retrospective study where records of seventeen consecutive patients with diagnosis of NF1, presenting Lisch nodules and choroidal alterations, and 17 age and gender-matched healthy control patients were evaluated. Fundus photographs, near infrared reflectance and enhanced depth imaging - optical coherence tomography images were reviewed. Retinal microvascular abnormalities and choroidal and retinal alterations in proximity of the retinal microvacular alterations were carefully noted.6 patients (35%) presented distinctive microvascular abnormalities. These consisted of small, tortuous vessels with a “spiral” or “corckscrew” aspect. They were second or third order, small tributaries of the superior or inferior temporal vein. These vessels were all located overlying choroidal alterations as observed with near infrared reflectance. Enhanced depth imaging - optical coherence tomography showed alteration of choroidal vasculature due to the presence of choroidal nodules but otherwise retinal and choroidal cross-sections were unremarkable for morphology.Retinal microvascular alterations overlying choroidal nodules in patients with NF1 can be considered another distinctive characteristic of the disease. Although the nature of these alterations is not clear, the authors speculate that functional disorders of vasomotor nerve cells, which originate in the embryonal neural crest can lead to their formation. Neurofibromatosis type 1 (NF1) is part of a group of heredofamilial disorders characterized by the presence of disseminated hamartoma for which Van der Hoeve introduced the termed “phakomatoses” in 1923 . In a unAll persons gave their informed consent prior to inclusion in the study. The research was given approval by the Institutional Review Board of the University of Rome “Sapienza” and was conducted in accordance with the tenets of the Declaration of Helsinki. Records of seventeen consecutive patients examined in 2014 with diagnosis of NF1 based on the stringent National Institutes of Health diagnostic criteria  were evaMicrovascular retinal abnormalities were identified in 6 patients (35%)  in a total of 17 patients with NF1 presenting Lisch and choroidal nodules. No similar alterations were identified in the healthy control patients. The microvascular alterations were localized in the vascular arcade area and were second or third order, small tributaries of the superior or inferior temporal vein. They consisted of small tortuous vessels with a “spiral” or “corkscrew” appearance in 3 female and 2 male patients (Figure NIR images showed that the retinal microvessels were all overlying patchy choroidal alterations. On cross-sectional EDI-OCT images these alterations corresponded to hyperreflective choroidal nodules which occupied space in the choroidal thickness and gave an irregular profile to the choroidal vasculature.Retinal angiomatosis in a patient with NF1 was reported in 1967 by Frenkel . The preIn the present study, all abnormal retinal microvessels were overlying patchy choroidal alterations as shown on NIR imaging. However, they were localized to the retina and did not involve the choroid, as shown by spectralis EDI-OCT cross-sections. Although choroidal vasculature is altered by the space occupying choroidal nodules, it is difficult to establish whether there is any correlation between these nodules and the abnormal microvascular retinal vessels.. The term “phakomatoses”, as described by Van der Hoeve, includes Recklinghausen’s neurofibromatosis, tuberous sclerosis, Hippel-Lindau disease, Sturge-Weber syndrome, Louis-Bar syndrome and Wyburn-Mason syndrome [Choroidal nodules are ovoidal bodies consisting of proliferating Schwann cells arranged in concentric rings around an axon . They shsyndrome . Even besyndrome , 12. Fursyndrome , 14. Thesyndrome . AlthougThis study describes “spiral” or “corkscrew” retinal microvascular alterations overlying choroidal nodules in patients with NF1. These alterations can be considered a distinctive retinal feature of NF1. Furthermore, as NF1 involves aberrant proliferation of multiple tissues of neural crest origin, these retinal alterations could possibly be related to functional disorders of vasomotor nerve cells.


Systemic use of epidermal growth factor receptor inhibitors (EGFRIs) has been shown to alter MHC expression and that of several chemokines, and to enhance immune cell recruitment into human skin. We hypothesized that EGFRIs may have value as cutaneous immune response modifiers, and determined the effects of topical application of an irreversible EGFRI on a well-established murine model of influenza vaccination. We found that a single topical application of an EGFRI led to increased levels of antibodies that inhibit influenza mediated hemagglutination and viral cytopathic effects. The topically applied EGFRI significantly enhanced the generation of vaccine-specific IL-4 and IFN-γ producing cells within skin-draining lymph nodes as early as one week following vaccination. The EGFRI/vaccine group showed a twelve-fold reduction in detectable pulmonary viral load four days after infection as compared to the vaccine alone control group. The reduction in the lung viral titers correlated with the survival rate, which demonstrated 100% protection in the EGFRI/vaccine immunized group but only 65% protection in the mice immunized with vaccine alone. These findings are significant because they demonstrate that inhibition of defined signaling pathways within the skin using small molecule kinase inhibitors provides a novel approach to enhance immune responses to vaccines. Since their widespread introduction in the 20Currently used adjuvants consist of compounds that are co-injected with vaccine antigens, and include a variety of aluminum salts, immunostimulatory molecules, and emulsions containing oil and waterThe delivery of vaccine components through the skin via intradermal, subcutaneous, and intramuscular injection are the most common routes of immunization. Despite the well characterized importance of the epidermis in regulating cutaneous immune responses, its relevance to vaccination has received little attention because it is largely bypassed during injection-based vaccinations. It is likely that as less invasive approaches of vaccination, such as those utilizing microneedles or nanoparticles become more widespread, the need to fully understand the role of the epidermis in the context of vaccination will gain importance67The pressing need to develop approaches to enhance the response to influenza vaccination is widely recognized11We selected an inhibitor of the EGFR for the following reasons. Prior studies have shown that EGFRI therapy is associated with increased recruitment of immune cells such as dendritic cells and macrophages into the skin1416in vitro inhibitory concentration 50 (IC50) values in the nanomolar range1415To determine the impact of a topical EGFRI on the response to influenza vaccination, we applied PD168393 prior to and at the site of intradermal (ID) delivery of H1N1 subunit influenza vaccine  . We sele+ and IgG+-secreting plasma cells in the skin draining lymph nodes and the spleen at one and two weeks post-vaccination. The antigen-specific IgM+ secreting plasma cells peaked one week after vaccination in both groups although at both time points, the EGFRI/vaccine group exhibited 2-fold higher plasma cell numbers than the vehicle/vaccine group (p < 0.001) (+-secreting plasma cells in the EGFRI/vaccine group peaked 2 weeks after vaccination reaching two-fold higher numbers than the vaccine/vehicle cohort (p < 0.001)  . The inf< 0.001) 20. No chTo assess the impact of the topically applied EGFRI on cytokine production and immune cell activation, we measured levels of IFN-γ and IL-4 producing influenza-specific lymphocytes in the skin-draining lymph nodes and spleens of vaccinated mice. Both IL-4 and IFN-γ producing influenza-specific cell numbers peaked as early as 7 days in the EGFRI/vaccine group reaching 3-fold and 5-fold differences respectively when compared to the vehicle/vaccine control numbers (p < 0.001) . The veh50 of homologous influenza virus at nine weeks following vaccination. Analysis of pulmonary viral loads revealed that the EGFRI/vaccine group had 12-fold and 14-fold lower titers than the vaccine alone group (p = 0.071) and vehicle/vaccine group respectively (p < 0.04)  . While tIn a separate cohort of vaccinated mice challenged with the same lethal dose, we observed that while all mice suffered weight losses with a maximum on day 7 post-challenge, the EGFRI/vaccine group exhibited the lowest losses among all groups (7%) and the vehicle/vaccine group suffered the highest losses (12%) . ApproxiTo determine how EGFR inhibition alters the expression of genes that are relevant to cutaneous immune responses in our model, we applied PD168393 or vehicle to the dorsum of BALB/c mice (5 mice per group) and measured steady state mRNA levels of several genes including those previously shown to be altered by EGFRI therapy. We selected a six-hour time point for these experiments because prior studies have shown that cellular responses to vaccination occur within hours and that PD168393 enhances the expression of the aforementioned chemokines at similar time pointsIn this study, we demonstrate that topical application of an EGFR kinase inhibitor can enhance the humoral and cellular response to influenza vaccination and augment protection against influenza challenge. These findings have several important implications. Firstly, to our knowledge, they provide the first proof-of-concept evidence that the response to vaccination can be modulated using a topically applied kinase inhibitor. In addition, our data also suggest that cellular events within regional lymph nodes that drain the skin can be modulated using this approach. While our study demonstrates the relevance of this approach in the context of an influenza vaccination, it may be more broadly applicable and relevant because at present there are few options available to pharmacologically modulate events within the lymph node basins draining epithelial tissues; sites that are critical during the early stages of antigen presentation and T cell activation against infections and malignancies.We selected an inhibitor of the EGFR pathway for these initial studies because prior studies have demonstrated that EGFRIs can enhance the elicitation phase of contact hypersensitivity and increase the expression of several chemokines such as CCL-2 (MCP-1), CXCL-10 (IP-10) and CCL27151324Many of the aforementioned effects of EGFRIs likely contribute to the unwanted skin rash that occurs in patients during systemic therapy for cancerAt the most basic level, the enormous efforts that have gone into the development of targeted therapies for the treatment of cancer have resulted in an expanding number of kinase inhibitors that are FDA approved for systemic use in humans. Thus, there is pre-existing information regarding the safety of these compounds at dosage levels that are much higher than would be needed for locally delivered applications. Likewise, in contrast to more conventional adjuvants where mechanistic details are largely lacking, there is detailed mechanistic information available for kinase inhibitors in terms of their molecular targets. Another potential advantage of kinase inhibitors stems from the fact that the size of these compounds is small relative to that of more conventional adjuvants or immunostimulatory molecules such as chemokines. As such, these molecules will likely be amenable to newer vaccination approaches such as aerosolized delivery systems and epicutaneous vaccination approaches. While our studies focused on an EGFR inhibitor, it is possible that targeting other kinases within the skin may have similar or even more robust effects on vaccination responses.Our study has several limitations that deserve mention. For example, while PD168393 is highly active against the EGFR, it is also active against other kinases such as ErbB-228293031Despite the aforementioned limitations, to our knowledge this is the first report in which a small molecule kinase inhibitor was re-purposed in this manner; namely as a vaccine adjuvant. While there is increasing recognition of the immune effects of targeted therapies such as EGFRIs, our data provide proof-of-concept evidence illustrating that some of these immune effects can be exploited locally to enhance the induction of antigen-specific immune responses. While the present study centers on the immune response to vaccination, this same approach may hold value to enhance local immune responses to infections and cancers of the skin and other tissues such as those of the mucous membranes that are accessible to locally delivered therapies.50) with the Reed-Munch formulaMadin-Darby canine kidney (MDCK) cells  were maintained in Dulbecco’s Modified Eagle’s Medium  containing 10% fetal bovine serum . Influenza virus stocks  were propagated in MDCK cells and purified by sucrose gradient centrifugation. The purity of the virus was determined by SDS PAGE followed by Coomassie blue staining50 (47 PFU) mouse adapted virus 9 weeks after vaccination and monitored for 14 days for body weight changes, fever, hunched posture, and mortality. Weight loss exceeding 25% was used as the experimental end point, at which mice were euthanized according to IACUC guidelines. All studies were approved by Emory University’s Institutional Animal Care and Use Committee.Six- to eight-week-old female BALB/c mice were purchased from Harlan Laboratories  and housed at Emory University Whitehead Animal Facility. Dorsal hair for intradermal vaccination was removed under systemic anesthesia with xylazine/ketamine cocktail. On the day of immunization 40 μl of 4 mM PD168393  or vehicle (DMSO and ethanol/negative control) were applied topically on shaved skin and allowed to dry for 10 min prior to intradermal injection of subunit influenza vaccine , 2 μg total protein in 50 μl PBS or PBS only  in the same area. Some mice received only a topical treatment and were not vaccinated as indicated in the text. Animals were bled (via submandibular puncture) at weeks 2, 4 and 8 after immunization and spleens, inguinal and axillary lymph nodes were collected at weeks 1 and 2 from euthanized mice for evaluation of cellular immune responses50/well of A/California/07/2009 virusInfluenza-specific hemagglutination inhibition titers (HAI) were assessed according to the WHO protocol6/200 μl cRPMI) were cultured for 36-48 h in the presence of 4 μg/ml A/California/07/09 influenza vaccine to enumerate vaccine-specific IL-4 and IFN-γ ELISPOT secreting cells. ELISPOT reagents were purchased from BD-PharMingen . Enumeration of vaccine-specific cytokine secreting cells was carried out by ELISPOT assay and counted using an ELISPOT reader and counter .Freshly isolated splenocytes and lymphocytes  or PD168393 (4 mM) to the backs of mice, mice were euthanized and the treated areas of skin excised and placed in RNAlater . Tissue samples were processed with the Qiagen miRNEAsy mini Kit 183 (Qiagen) and TissueLyser II bead mill  according to the manufacturers’ protocols. Total RNA was quantitated on a ThermoScientfic NanoDrop 1000 and integrity assessed on an Agilent 2100 Bioanalyzer using the Agilent RNA 6000 Nano Kit . 10 μg of total RNA was converted to cDNA using the Applied Biosystems  High-Capacity cDNA Archive Kit according to the manufacturer’s protocol.Real Time Primers LLC, Elkins Park, PA and sequences are available upon request.Quantitative real-time PCR was performed on a Bio-Rad CFX96 thermocycler  and normalized expression levels calculated using the ΔΔCt mode of CFX Manager Software . Levels of steady state mRNA of the following genes was assessed: chemokine (C-C motif) ligand (CCL) 2, CCL5, MHC class II transactivator (CIITA), chemokine (C-X-C motif) ligand (CXCL) 10, epidermal growth factor receptor (EGFR), murine MHC class I and II genes , interferon regulatory factor (IRF) 1, NLR family CARD domain containing (NLRC) 5. Steady state mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase (HRPT) 1 and peptidylprolyl isomerase A (PPIA) were also measured. All primers were purchased from Twenty four and 48 hours following application of PD168393 or vehicle, mice were euthanized and dorsum skin tissue samples excised and fixed in buffered formalin solution, embedded in paraffin, sectioned, and the slides stained with hematoxylin and eosin. The stained slides were scanned and reviewed digitally as well as using light microscopy.oC until further use. Five micron skin cryosections were fixed in cold acetone and incubated with PerCP anti-mouse I-A/I-E clone M5/114.15.2  diluted in PBS with 2% bovine serum albumin, followed by VECTASHIELD mounting medium with DAPI  to stain the nuclei. The stained samples were examined under fluorescence microscopy.Mouse ears were collected 24 and 48 hours after topical application of EGFRI or vehicle in Tissue-Tek® O.C.T. Compound,  and stored at −80 t-test and a one-way ANOVA including Bonferroni’s multiple comparison test were utilized. Two-way ANOVA test was used for the cell-based immune responses. For lung titers and body weight changes we applied two-tailed unpaired Student’s t-test. For the survival curves, statistics were calculated using a Log-rank (Mantel-Cox) test. A p-value less than 0.05 was considered significant. Means and standard deviations were calculated from quadruplicate runs and at least two independent animal experiments. The statistical significance of differences for real-time PCR data was calculated using a two-tailed unpaired Student’s t-test for normally distributed data and a Mann-Whitney test when data was not normally distributed as determined by the method of Komolgorov and Smirnov.For humoral immune responses (HAI and NT) a two-tailed unpaired Student’s How to cite this article: Pulit-Penaloza, J. A. et al. Modulation of influenza vaccine immune response using an epidermal growth factor receptor kinase inhibitor. Sci. Rep. 5, 12321; doi: 10.1038/srep12321 (2015).


Breast cancers produce cytokines and chemokines that attract and polarize immune cells in a manner that promotes disease progression and metastasis. For example, macrophages are polarized to anti-inflammatory type II tumor activating macrophages (TAMs), and CD4Studies in mice and humans show that Tregs also are developed when inhibiting the kinase mTOR  via a poorly understood mechanism. mTOR forms two complexes in the cell, mTORC1 and mTORC2, which regulate multiple metabolic processes. In particular, mTORC1 is inhibited by the immunosuppressant rapamycin and stimulates the translation of mRNAs involved in cell growth and proliferation. Protein synthesis is a highly regulated process involving general mRNA and selective mRNA translation. Our hypothesis is that mTOR downregulation alters lymphocyte gene expression by favoring the translation of specific mRNAs required for Treg differentiation.We generated human Tregs induced in culture (iTregs) by mTORC1 inhibition through RAD001 (everolimus), a rapalog used as an anti-cancer drug. By contrast, mTORC1/2 dual inhibitor PP242 blocked the growth of all the lymphocytes, pointing to a specific role of mTORC1 in Treg differentiation. In fact, we found that the proliferation of highly suppressive iTregs requires co-treatment with RAD001 and the cytokine TGFβ. Murine models do not require TGFβ, suggesting that caution should be used in extending findings from mouse to human Treg studies. Protein synthesis analysis in double-treated (RAD001+TGFβ), control-treated, RAD001- and TGFβ-treated cells shows that translation is greatly inhibited in the double-treated cells. Genome-wide translation profiling of mRNAs associated with actively translating ribosomes confirmed that only a selective pool of specific mRNAs is translated in the iTreg population. These specific mRNAs may be recruited to ribosome via an mTOR-independent mechanism involving PAIP2, and eIF4G adapter protein, and the ribosomal protein S25.Our work indicates the importance of selective translational regulation as an additional determinant of gene expression regulating T cell fate. We suggest that iTreg development is facilitated by selective translation of specific mRNAs whose recruitment to the ribosome is augmented when mTORC1 is inhibited. These findings also suggest that cancer treatments causing an improper balance of mTORC1 inhibition might attenuate the anti-tumor immune response through development of Tregs in the tumor microenvironment


These data are compared to biochemical and functional measures of synaptogenesis.Synaptogenesis is a critical neurodevelopmental process whereby pre- and postsynaptic neurons form apposed sites of contact specialized for chemical neurotransmission. Many neurodevelopmental disorders are thought to reflect altered patterns of synaptic connectivity, including imbalances between excitatory and inhibitory synapses. Developing rapid throughput approaches for assessing synaptogenesis will facilitate toxicologic and drug screening studies of neurodevelopmental disorders. The current study describes the use of high-content imaging to quantify the ontogeny of excitatory and inhibitory synapses using in vitro (DIV). As determined by ELISA, the increase in synaptophysin expression levels as cultures matured was similar between hippocampal and cortical cultures. High-content imaging of immunoreactivity of excitatory and inhibitory synaptic biomarkers demonstrated an overall greater number of synapses in hippocampal relative to cortical neurons with marked differences in the pattern of inhibitory synapse development between these two neuronal cell types. Functional assays revealed that both the mean firing rates and mean bursting rates were significantly increased in cortical cultures relative to hippocampal cultures. This difference may reflect decreased inhibitory synaptic tone in cortical versus hippocampal cultures.The ontogenetic patterns of synapse formation were compared between primary rodent hippocampal and cortical neurons over 28 days versus inhibitory synapses. The functional significance of differences in the balance of excitatory to inhibitory synapses was confirmed by the assessment of network activity using microelectrode arrays. These results suggest that high-content imaging and microelectrode arrays provide complementary approaches for quantitative assessment of synaptogenesis, which should provide a robust readout of toxicologic and pharmacologic effects on this critical neurodevelopmental event.These data demonstrate differences and similarities in the ontogeny of synaptogenesis between hippocampal and cortical neurons, depending on the biological level examined. Assessment of synaptophysin protein levels by ELISA showed a general increase in synapse formation in both cell types with increasing time in culture, while high-content imaging was able to delineate cell type-dependent differences in formation of excitatory  Synapses can be either excitatory  or inhibitory . The balance between excitatory and inhibitory neurotransmission is critical for proper development and function of the central nervous system . AbnormaExcitatory and inhibitory synapses are distinguished by the type of neurotransmitter that is released from the presynaptic terminal and by the profile of pre- and postsynaptic proteins within the synapse. For example, in the mature central nervous system (CNS), glutamatergic synapses are excitatory and are characterized by the release of glutamate from the presynaptic terminal, the presence of vesicular glutamate transporter 1 (vGLUT1) in the presynaptic vesicle pool and the presence of postsynaptic density 95 (PSD95) in the postsynaptic density ,5. In coin vitro, including quantification of synaptic protein levels using antibody-based methods  and functional assessment using electrophysiology [in vitro at the cellular level based on immunocytochemical localization of synaptic proteins and to detect chemical-induced neurotoxicity [in vitro [A number of methods have been used to measure synaptogenesis ysiology -9. Howevtoxicity ,11. Signin vitro .in vitro models of neurodevelopment: primary cultures of rat cortical and hippocampal neurons. Synapse formation was measured over 28 DIV at differing levels of biological complexity: 1) at the molecular level using ELISA to quantify the level of synaptophysin protein; 2) at the cellular level using HCI to quantify the immunoreactivity of excitatory and inhibitory synaptic biomarkers; and 3) at the functional level using MEA recordings. Our data demonstrate quantitative and qualitative similarities and differences in measures of synaptogenesis across methods and cell types.In the present study, we examined the ontogeny of synaptogenesis in two widely used 2) did not contain a sufficient number of viable neurons to examine excitatory and inhibitory synaptogenesis by HCI at time points past 7 DIV. Therefore, the plating density for cortical neurons was increased for both ELISA and HCI experiments. Likewise, cortical and hippocampal cultures seeded on MEAs at densities comparable to those used in HCI experiments did not develop sufficient levels of synaptic activity to enable reliable quantification. Thus, we adopted a plating method in which cells are restricted to a drop of medium centered on the MEA, which had previously proven to reliably support the development of synaptic network activity [Ideally, the same plating density of cortical and hippocampal neurons would be used across all the biological levels of synaptogenesis examined; however, results of pilot experiments (data not shown) indicated that this strategy was not feasible. Rather, plating densities needed to be optimized for each cell type in each platform. For example, the basal rate of neuronal cell loss in 96-well cultures was significantly greater in cortical cultures relative to hippocampal cultures. Cortical cultures plated at the same density as hippocampal cultures  in both hippocampal and cortical cultures  was observed with no main effect of cell type or interaction. Post hoc analysis demonstrated that synaptophysin levels at 21 and 28 DIV were significantly increased relative to earlier time points.Data generated using a synaptophysin-specific ELISA normalized to total protein, similarly demonstrates increasing levels of synaptophysin with increasing time s Figure B. SynaptTo qualitatively assess whether the synaptophysin immunoreactivity recognized both excitatory and inhibitory synapses, we co-labeled rat hippocampal and cortical cultures for synaptophysin, vGLUT1 and vGAT, which are expressed in the presynaptic terminals of excitatory and inhibitory synapses, respectively -6. As deversus inhibitory synapses, we used HCI analysis of hippocampal and cortical cultures immunostained for the synaptic vesicle-associated proteins vGLUT1 and vGAT. Hippocampal and cortical neurons at 21 DIV were immunolabeled with antibodies specific for microtubule-associated protein 2 (MAP2) to identify dendrites (blue), vGLUT1 to mark excitatory synapses (red) and vGAT to distinguish inhibitory synapses (green) . Post-hoc analysis demonstrated that the number of cortical neurons per field was greater than the number of hippocampal neurons per field at each time point  indicating that the time course of dendrite outgrowth was different between the two cell types. Post-hoc analysis demonstrated that total dendrite length was greater in hippocampal neurons relative to cortical neurons at each time point  was observed for the total number of excitatory synapses per neuron   Figure A. The to3,81 = 17.86, p < 0.0001) was also observed for the total number inhibitory synapses per neuron   Figure B. Simila3,81 = 3.67, p = 0.0155) was observed for the number of excitatory synapses per cell body  was observed for the number of inhibitory synapses per cell body  was observed for the number of excitatory synapses per dendrite length  was also observed for the number of inhibitory synapses per dendrite length . Values above 0 indicate that more excitatory than inhibitory synapses whereas values below 0 indicate the opposite, e.g., inhibitory synapses are more numerous than excitatory synapses. In hippocampal neurons, vGLUT1 immunopositive synapses were more numerous than vGAT immunopositive synapses in the dendrite compartment at all times examined. In contrast, vGAT synapses became more numerous than vGLUT1 synapses in the cell body compartment at time points beyond 7 DIV  was observed for the number of active channels  was observed for mean firing rate  was also observed for mean burst rate .in vivo, and as a biomarker of synaptogenesis in cultured hippocampal neurons [versus hippocampal neurons cultured from embryonic mice [Synaptophysin is an integral membrane protein of synaptic vesicles whose expression at both the transcript and protein level increases as neurons mature ,17. Thus neurons ,19. Usin neurons ,20. Simi neurons . Qualitanic mice . HoweverOur findings are consistent with the literature that the expression of synaptophysin correlates well with neuronal maturation; however, because not all synaptic vesicles are localized to synapses, the immunocytochemical localization of synaptophysin does not necessarily indicate a true synapse . Furtherin vitro dendritic growth between the two cell types may contribute to this effect.The development of the dendritic arbor is intimately tied to synapse formation. Synaptic connections increase in parallel to dendritic development, and abnormalities in dendritic length are associated with changes in synapse number and function -29. In oin vivo observations that rates of synaptogenesis differ between and within brain regions even at similar stages of brain development [in vitro, lower cell densities result in faster development of synapses and higher synapse-to-neuron ratios [The numbers of vGLUT1 and vGAT immunopositive puncta also increased in hippocampal and cortical cell cultures with increasing time in culture, which is consistent with previous experiments performed in our lab  and withelopment ,33. Anotelopment -36. Thiselopment . Howevern ratios . This con ratios .versus cortical cell cultures. In general, there were significantly more excitatory and inhibitory synapses formed in hippocampal cell cultures relative to cortical cell cultures beginning on DIV 14 through DIV 28. With regards to the ratio of excitatory to inhibitory synapses, previous studies of hippocampal neurons have reported that the number of excitatory synapses generally is greater than the number of inhibitory synapses in mature neuronal cell populations [versus inhibitory synapses and the ratio of excitatory to inhibitory synapses that form on MAP2 immunopositive dendrites. The one notable exception to this generalization was the ratio of excitatory to inhibitory synapses formed on neuronal cell bodies in hippocampal cell cultures. The number of vGAT immunopositive puncta formed on neuronal cell bodies was significantly higher in hippocampal versus cortical neurons, and the ratio between these two synaptic types was such that vGAT immunopositive puncta outnumbered vGLUT1 immunopositive puncta at DIV 14 through 28. Inhibitory synapse input predominates in pyramidal cell somata and it has been speculated this is to match the inhibitory efficacy in dendrites, due to the relatively larger diameter of the pyramidal cell somata [In addition to cell type-specific differences in the ontogeny of synaptogenesis, there was a significant difference in the number and ratio of excitatory and inhibitory synapses in hippocampal ulations ,41. Our l somata . Thus, tl somata .versus inhibitory synapses is predictive of network activity, we also measured synaptic function by recording activity in hippocampal and cortical cultures plated onto MEAs. MEAs enable simultaneous, noninvasive extracellular recording over long periods of time in a relatively high-throughput format compared to traditional electrophysiological techniques. Cultured neurons can be followed from the time of isolation until the development of spontaneous firing, providing a unique opportunity to record the ontogeny of neuronal network activity. These measurements have a wide variety of applications, from basic research to drug discovery and toxicology screening [2). Culture density has consistently been shown to have significant influence on the development, localization, and function of synapses, with higher density cultures exhibiting earlier developmental onset of network activity [The relative numbers of excitatory and inhibitory synapses are a critical determinant of network activity, which is the functional readout of synaptogenesis. To determine whether the data we obtained from high content imaging of excitatory creening ,43,44. Wcreening ,46, studcreening ,48. In bcreening ,49. A poactivity ,50,51. Tactivity  .in vitro excitatory connections [versus cortical cultures, as all of these factors can influence neuronal network activity [Interestingly, the ontogeny of network activity was similar between the two culture models; however, at DIV 14, cortical cultures displayed significantly higher firing rates and burst activity than hippocampal neurons. This difference may reflect the higher vGLUT1/vGAT ratio observed in the cell body compartment of cortical neurons relative to hippocampal neurons. Inhibitory GABAergic input modulates firing behavior such that decreased GABAergic neurotransmission shifts firing to a bursting pattern ,54, and nections ,57. Howeactivity .in vitro synaptogenesis between hippocampal and cortical neuronal cell cultures. Hippocampal neurons were observed to mature faster than cortical cell cultures, as evidenced by increased dendritic lengths and numbers of vGLUT1 and vGAT immunopositive puncta at any given DIV. Another marked difference between the two culture models was the decreased excitatory to inhibitory synaptic input to the cell soma in hippocampal neurons versus cortical neurons. Cortical neurons in general exhibited a slower developmental timeline, showing more robust increases at later time points. Indeed, it appeared that dendritic length and vGLUT1/vGAT expression had not yet plateaued in cortical cell cultures by the end of the experimental period. At the level of network activity, hippocampal and cortical cells displayed remarkable similarity in the ontogeny of firing rates; however, cortical neurons exhibited significantly higher burst activity at DIV 14, which correlated with spatiotemporal differences in the relative ratio of excitatory to inhibitory synapses between the two culture models. Collectively, our data indicate that the ontogeny of in vitro synaptogenesis observed in the present study is largely consistent with data reported in previous studies and that cell type-dependent differences exist in synaptogenic profiles.In summary, these data provide a comparison of the ontogeny of These results further suggest that utilization of multiple techniques can provide a more integrative view of synaptogenesis, which may provide more useful and predictive insight of how chemical exposures and pharmacologic interventions influence this critical neurodevelopmental endpoint. Synaptophysin ELISA may be useful for assessing maturation of cultures but is not very useful for providing detailed information about synapse types or functionality. HCI can provide quantitative information regarding the types of synapses being formed and their subcellular distribution but does not provide information regarding synaptic function. MEAs provide measurements of synaptic activity, but changes in underlying cellular events that influence activity measurements  cannot be determined. Our findings demonstrate that high-throughput immunocytochemical assays to measure excitatory and inhibitory synapse formation, in parallel with functional studies of neuronal activity using MEA recordings, can be used to study the effect of environmental chemicals and/or drugs on synaptic network formation and function.ad libitum.Animals were treated humanely and with regard for alleviation of suffering according to protocols approved by the Institutional Animal Care and Use Committee of the University of California, Davis. Timed pregnant Sprague Dawley rats were purchased from Charles River Laboratory  and individually housed in standard plastic shoe box cages with corn cob bedding in a temperature (22 ± 2°C) controlled room on a 12 h reverse light-dark cycle. Food and water were provided 2 while hippocampal neurons were plated at a density of 31,250 cells/cm2. For analysis of network activity, dissociated cells were plated on 12-well polystyrene MEA plates in which each well contained 64 nanoporous platinum electrodes . MEAs were precoated with poly-L-lysine  and laminin  and cells were plated at a density of 235,785 cells/cm2. All cultures were maintained in Neurobasal-A (Invitrogen) supplemented with 2% B27 (Invitrogen) and 2 mM Glutamax (Invitrogen). At DIV 4, cytosine-D-arabinofuranoside (Sigma) was added to the medium at a final concentration of 5 μM. Half of the medium was replaced with fresh Neurobasal-A supplemented with B27 once weekly.Primary cultures of dissociated cortical and hippocampal cells were prepared and maintained as previously described . BrieflyThe relative levels of synaptophysin were quantified in primary cultures of cortical and hippocampal cells plated on 6-well plates and maintained as described above. Cell lysates were collected at 7, 14, 21 and 28 DIV in 150 μL of RIPA buffer  containing HALT protease and phosphatase inhibitors . Samples were centrifuged and then stored at -80°C. Sandwich ELISAs using antibodies specific for synaptophysin were adapted from a previously described method . BrieflyCortical and hippocampal cell cultures were fixed in 4% paraformaldehyde containing 4% sucrose for 1 h. Cultures were incubated with Hoechst Stain 33258 (3 μg/mL) (Invitrogen) diluted in phosphate buffered saline  for 5 min then rinsed with PBS and permeabilized in 0.2% Triton X-100 in PBS for 5 min. Cultures were then blocked with 5% bovine serum albumin in PBS for 20 min and incubated with primary antibodies overnight at 4°C. Primary antibody solutions included guinea pig anti-MAP2 , rabbit anti-vGLUT1 , mouse anti-vGAT , rabbit anti-synapsin  or mouse anti-synaptophysin . After extensive rinsing with PBS, cultures were incubated with Alexa-Fluor® secondary antibodies  for 2 h at room temperature. After washing with PBS to remove unbound secondary antibody, wells were filled with PBS at 4°C, sealed tightly with Parafilm®  and shipped to the U.S.E.P.A. laboratories in Research Triangle Park, NC under refrigeration.TI (Thermo Scientific) as described previously [Image acquisition and analysis were performed using a Cellomics® ArrayScan® Veviously . Images Image analysis was performed using the Cellomics® Neural Profiling BioApplication. A previously developed high-content image analysis algorithm  was optiversus dendrites as well as total synapses (cell body + dendrites).The total number of vGLUT1 and vGAT puncta was categorized as either contacting the cell body  or contacting a dendrite . Endpoints analyzed in this study included: 1) the number of neurons per field; 2) dendrite length per neuron; 3) the number of vGLUT1 puncta per neuronal cell body; 4) the number of vGAT puncta per neuronal cell body; 5) the number of vGLUT1 puncta per μm dendrite length; 6) the number of vGAT puncta per μm dendrite length; 7) the total number of vGLUT1 puncta per neuron; and 8) the total number vGAT puncta per neuron. For each synaptic type, the total number of synaptic puncta includes both cell body- and dendrite-localized synapses. The ratio of excitatory to inhibitory (vGLUT1/vGAT) synapses was also calculated with respect to the number localized to cell bodies Spontaneous network activity was recorded from the MEAs as previously described . Data weStatistical analyses were performed using GraphPad Prism software . Specific statistical tests are identified in the results section and in figure legends.