Cell culture process development: advances in process engineering

Representatives from the cell culture process development community met on September 11 and 12, 2006 at the ACS National Meeting in San Francisco to discuss "Cell Culture Process Development: Advances in Process Engineering". This oral session was held as part of the Division of Biochemical Technology (BIOT) program. The presentations addressed the very small scale (less than 1 mL) to the very large scale (20,000 L). The topics covered included development of high throughput cell culture screening systems, modeling and characterization of bioreactor environments from mixing and shear perspectives at both small and large scales, systematic approaches for improving scale-up and scale-down activities, development of disposable bioreactor technologies, and novel perfusion culture approaches. All told, this well-attended session resulted in a valuable exchange of technical information and demonstrated a high level of interest within the process development community.     

Skin-derived dendritic cells can mediate deletional tolerance of class I-restricted self-reactive T cells

Skin-draining lymph nodes contain a number of dendritic cell (DC) subsets of different origins. Some of these are migratory, such as the skin-derived epidermal Langerhans cells and a separate dermal DC subset, whereas others are lymphoid resident in nature, such as the CD8+ DCs found throughout the lymphoid tissues. In this study, we examine the DC subset presentation of skin-derived self-Ag by migratory and lymphoid-resident DCs, both in the steady state and under conditions of local skin infection. We show that presentation of self-Ag is confined to skin-derived migrating DCs in both settings. Steady state presentation resulted in deletional T cell tolerance despite these DCs expressing a relatively mature phenotype as measured by traditional markers such as the level of MHC class II and CD86 expression. Thus, self-Ag can be carried to the draining lymph nodes by skin-derived DCs and there presented by these same cells for tolerization of the circulating T cell pool.     

Spontaneous autoreactive memory B cell formation driven by a high frequency of autoreactive CD4+ T cells

Although somatically mutated autoantibodies are characteristic of many autoimmune diseases, the processes that can lead to their development remain poorly understood. We have examined the formation of autoreactive memory B cells in PevHA mice, which express the influenza virus PR8 hemagglutinin (HA) as a transgenic membrane bound neo-self-Ag. Using a virus immunization strategy, we show that PR8 HA-specific memory B cell formation can occur in PevHA mice, even though a major subset of PR8 HA-specific B cells is negatively selected from the primary repertoire. Moreover, PR8 HA-specific memory B cells develop spontaneously in TS1 x PevHA mice, which coexpress a transgenic PR8 HA-specific TCR and contain a high frequency of HA-specific CD4(+) T cells. Notably, autoreactive memory B cell formation occurred in TS1 x PevHA mice even though approximately half of the HA-specific CD4(+) T cells were CD25(+)Foxp3(+) cells that could significantly attenuate, but did not completely abolish HA-specific autoantibody production in an adoptive transfer setting. The findings provide evidence that a high frequency of autoreactive CD4(+) T cells can be sufficient to promote autoreactive memory B cell formation in the absence of signals provided by overt immunization or infection and despite the presence of abundant autoantigen-specific CD4(+)CD25(+)Foxp3(+) regulatory T cells.     

Environment-specific heterozygote deficiency and developmental instability in hybrid Mytilus

The multiple discrete hybrid zones that characterize Mytilus blue mussels allow a novel, non-manipulative, examination of the selective pressures that create and maintain species. If endogenous genetic incompatibility is solely responsible for post-zygotic isolation, then individuals of a specified hybrid genotype are expected to show similar average fitness across environments. However, if hybrid fitness differs across environments, then exogenous selection is implicated, either via ecological selection or environment-specific expression of intrinsic genetic incompatibilities. Correspondence between developmental instability of hybrids and heterozygote deficiency, estimated in two M. trossulus×M. galloprovincialis hybrid zones on the coast of North America, indicates that environment-dependent selection against hybrids may contribute to reproductive isolation among Pacific Mytilus species.     

Novel Crohn disease locus identified by genome-wide association maps to a gene desert on 5p13.1 and modulates expression of PTGER4

To identify novel susceptibility loci for Crohn disease (CD), we undertook a genome-wide association study with more than 300,000 SNPs characterized in 547 patients and 928 controls. We found three chromosome regions that provided evidence of disease association with p-values between 10⁻⁶ and 10⁻⁹. Two of these (IL23R on Chromosome 1 and CARD15 on Chromosome 16) correspond to genes previously reported to be associated with CD. In addition, a 250-kb region of Chromosome 5p13.1 was found to contain multiple markers with strongly suggestive evidence of disease association (including four markers with p < 10⁻⁷). We replicated the results for 5p13.1 by studying 1,266 additional CD patients, 559 additional controls, and 428 trios. Significant evidence of association (p < 4 × 10⁻⁴) was found in case/control comparisons with the replication data, while associated alleles were over-transmitted to affected offspring (p < 0.05), thus confirming that the 5p13.1 locus contributes to CD susceptibility. The CD-associated 250-kb region was saturated with 111 SNP markers. Haplotype analysis supports a complex locus architecture with multiple variants contributing to disease susceptibility. The novel 5p13.1 CD locus is contained within a 1.25-Mb gene desert. We present evidence that disease-associated alleles correlate with quantitative expression levels of the prostaglandin receptor EP4, PTGER4, the gene that resides closest to the associated region. Our results identify a major new susceptibility locus for CD, and suggest that genetic variants associated with disease risk at this locus could modulate cis-acting regulatory elements of PTGER4.     

Experimental drought and heat can delay phenological development and reduce foliar and shoot growth in semiarid trees

Higher temperatures associated with climate change are anticipated to trigger an earlier start to the growing season, which could increase the terrestrial C sink strength. Greater variability in the amount and timing of precipitation is also expected with higher temperatures, bringing increased drought stress to many ecosystems. We experimentally assessed the effects of higher temperature and drought on the foliar phenology and shoot growth of mature trees of two semiarid conifer species. We exposed field‐grown trees to a ~45% reduction in precipitation with a rain‐out structure (‘drought’), a ~4.8 °C temperature increase with open‐top chambers (‘heat’), and a combination of both simultaneously (‘drought + heat’). Over the 2013 growing season, drought, heat, and drought + heat treatments reduced shoot and needle growth in piñon pine (Pinus edulis) by ≥39%, while juniper (Juniperus monosperma) had low growth and little response to these treatments. Needle emergence on primary axis branches of piñon pine was delayed in heat, drought, and drought + heat treatments by 19–57 days, while secondary axis branches were less likely to produce needles in the heat treatment, and produced no needles at all in the drought + heat treatment. Growth of shoots and needles, and the timing of needle emergence correlated inversely with xylem water tension and positively with nonstructural carbohydrate concentrations. Our findings demonstrate the potential for delayed phenological development and reduced growth with higher temperatures and drought in tree species that are vulnerable to drought and reveal potential mechanistic links to physiological stress responses. Climate change projections of an earlier and longer growing season with higher temperatures, and consequent increases in terrestrial C sink strength, may be incorrect for regions where plants will face increased drought stress with climate change.     

Inferring processes of coevolutionary diversification in a community of Panamanian strangler figs and associated pollinating wasps*

The fig and pollinator wasp obligate mutualism is diverse (～750 described species), ecologically important, and ancient (～80 Ma). Once thought to be an example of strict one‐to‐one cospeciation, current thinking suggests genera of pollinator wasps codiversify with corresponding sections of figs, but the degree to which cospeciation or other processes contribute to the association at finer scales is unclear. Here, we use genome‐wide sequence data from a community of Panamanian strangler figs and associated wasp pollinators to estimate the relative contributions of four evolutionary processes generating cophylogenetic patterns in this mutualism: cospeciation, host switching, pollinator speciation, and pollinator extinction. Using a model‐based approach adapted from the study of gene family evolution, our results demonstrate the importance of host switching of pollinator wasps at this fine phylogenetic and regional scale. Although we estimate a modest amount of cospeciation, simulations reveal the number of putative cospeciation events to be consistent with what would be expected by chance. Additionally, model selection tests identify host switching as a critical parameter for explaining cophylogenetic patterns in this system. Our study demonstrates a promising approach through which the history of evolutionary association between interacting lineages can be rigorously modeled and tested in a probabilistic phylogenetic framework.