The Spatial and Temporal Expression of Polymeric Immunoglobulin Receptor (pIgR) in Zebrafish Larvae

The mucosal immune system (a branch of the adaptive immune system) mediates infections in the body?s mucosal surfaces, where the organism\u27s internal structures come into contact with the external environment (i.e. the gastrointestinal tract). Polymeric immunoglobulin receptor (pIgR) is essential for the proper functioning of the mucosal immune system. pIgR transports secretory immunoglobulins across intestinal epithelial cells into the lumen, the site of the pathogens. Along with this function, certain aspects of pIgR\u27s sequence and structure have been evolutionarily conserved in many classes of vertebrates. Zebrafish (Danio rerio) often serve as key model organisms in studies of vertebrates, as they are small, easy to raise, and virtually translucent as embryos/larvae. It is known that the zebrafish adaptive immune system does not become fully functional until approximately four weeks post-fertilization. However, indicators of adaptive immune system activation (including the expression of the recombination activating genes) demonstrate that the adaptive immune system may begin to function as early as four days post-fertilization. The spatial and temporal expression pattern of pIgR in zebrafish larvae is not yet known. It is expected that pIgR will begin to be expressed at approximately four days post-fertilization in areas surrounding the mucosal surfaces. Protocols, including in-situ hybridizations and reverse-transcriptase polymerase chain reaction (RT-PCR), will be utilized in order to assess the expression pattern of pIgR in zebrafish larvae. Characterizing this expression pattern will provide profound implications for the evolution of the adaptive immune system as well as of pIgR in particular

How Specific Should Immunological Memory Be?

Protection against infection hinges on a close interplay between the innate immune system and the adaptive immune system. Depending on the type and context of a pathogen, the innate system instructs the adaptive immune system to induce an appropriate immune response. Here, we hypothesize that the adaptive immune system stores these instructions by changing from a naive to an appropriate memory phenotype. In a secondary immune reaction, memory lymphocytes adhere to their instructed phenotype. Because cross-reactions with unrelated Ags can be detrimental, such a qualitative form of memory requires a sufficient degree of specificity of the adaptive immune system. For example, lymphocytes instructed to clear a particular pathogen may cause autoimmunity when cross-reacting with ignored self molecules. Alternatively, memory cells may induce an immune response of the wrong mode when cross-reacting with subsequent pathogens. To maximize the likelihood of responding to a wide variety of pathogens, it is also required that the immune system be sufficiently cross-reactive. By means of a probabilistic model, we show that these conflicting requirements are met optimally by a highly specific memory lymphocyte repertoire. This explains why the lymphocyte system that was built on a preserved functional innate immune system has such a high degree of specificity. Our analysis suggests that 1) memory lymphocytes should be more specific than naive lymphocytes and 2) species with small lymphocyte repertoires should be more vulnerable to both infection and autoimmune diseases

The role of idiotypic interactions in the adaptive immune system: a belief-propagation approach

In this work we use belief-propagation techniques to study the equilibrium behaviour of a minimal model for the immune system comprising interacting T and B clones. We investigate the effect of the so-called idiotypic interactions among complementary B clones on the system's activation. Our result shows that B-B interactions increase the system's resilience to noise, making clonal activation more stable, while increasing the cross-talk between different clones. We derive analytically the noise level at which a B clone gets activated, in the absence of cross-talk, and find that this increases with the strength of idiotypic interactions and with the number of T cells signalling the B clone. We also derive, analytically and numerically, via population dynamics, the critical line where clonal cross-talk arises. Our approach allows us to derive the B clone size distribution, which can be experimentally measured and gives important information about the adaptive immune system response to antigens and vaccination.Comment: 37 pages, 18 figure

MicroRNA regulation of CD8+ T cell responses.

MicroRNAs (miRNAs) are a class of short noncoding RNAs that play critical roles in the regulation of a broad range of biological processes. Like transcription factors, miRNAs exert their effects by modulating the expression of networks of genes that operate in common or convergent pathways. CD8+ T cells are critical agents of the adaptive immune system that provide protection from infection and cancer. Here, we review the important roles of miRNAs in the regulation of CD8+ T cell biology and provide perspectives on the broader emerging principles of miRNA function

CRISPR-mediated adaptive immune system in E. coli

CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) genes comprise an RNA-guided adaptive immune system in prokaryotes. Cas proteins can acquire short fragments, called spacers, from the invader DNA or RNA and integrate these spacers into the host genomic CRISPR locus. Once transcribed and processed to short CRISPR RNAs (crRNA), the crRNA spacers can guide Cas surveillance complexes to DNA and/or RNA target sequences, called protospacers, resulting in CRISPR interference through target cleavage. In the type I CRISPR system, an existing protospacer can accelerate new spacer acquisition from the same invader DNA, a process called priming. We have investigated the immune mechanism of the type I-E CRISPR-Cas system in Escherichia coli K12. The research presented in this dissertation has focused on how Cascade searches DNA to locate the target and how crRNA sequence and Cascade conformation control interference and priming activities. The Cascade surveillance complex must locate targets rapidly to ensure timely immune response, but the mechanism of this search process remains unclear. We developed a single-molecule fluorescence resonance energy transfer (FRET) assay to directly visualize the Type I-E Cascade surveillance complex searching DNA in real time. We find that Cascade randomly samples DNA through short-lived nonspecific electrostatic contacts and quickly dissociates from dsDNA. Cascade locates its target by first searching for short recognition sequences called protospacer adjacent motifs (PAMs). We find that Cascade dwells longer at PAM sites based on interaction with the PAM recognition motif and a lysine-rich loop in Cse1. In addition, we also identify a motif in the Cas7 backbone subunit that is essential for the searching process. Our findings provide a comprehensive structural and kinetic model for efficient target searching by Cascade. Once Cascade locates its target, it recruits the trans-acting nuclease Cas3 to trigger CRISPR interference. However, mutations in the PAM or the PAM-proximal region of the protospacer, termed the seed, can block interference and lead to primed adaptation. The importance of the seed region and PAM motif has been studied using a few spacers in Type I-E CRISPR system in E. coli K12. However, it is unknown whether spacer sequence has an effect on the activities of CRISPR system. We have analyzed CRISPR interference and priming using 18 endogenous spacers in E. coli K12 to reexamine the PAM and seed sequence requirements and found that CRISPR interference and priming are strongly influenced by spacer sequence. Our interference data for these 18 spacers also indicate that CRISPR interference is far more tolerant of mutations in the seed and the PAM than previously reported. We further analyzed spacer sequence-specific tolerance of seed or PAM mutations. Our results indicate that seed and PAM mutational tolerance are highly dependent on spacer sequence. We further show that cytosine residue at the -3 and -2 position of the PAM abolishes both interference and priming, indicating that CRISPR-Cas systems avoid self-spacer targeting by avoiding the final 3 nucleotides of the repeat (CCG). In summary, our findings show that CRISPR activities strongly depend on the spacer sequence and CRISPR-Cas systems avoid self-spacers targeting by avoiding recognize the repeat (CCG). Our studies reveal that some spacer sequences may more readily overcome immune system evasion through invader evolution. Surprisingly, some PAM mutations have little effect on the equilibrium binding affinity of Cascade, but these mutations still block CRISPR interference. We and other groups have found that Cas3 cannot degrade the Cascade-bound target, suggesting that Cascade may adopt an alternative conformation that blocks Cas3 recruitment when bound to these targets. To test this hypothesis, we developed a novel FRET system to study the conformational dynamics of the Cse1 subunit of Cascade, which recognizes the PAM and recruits Cas3. Our results reveal that Cascade adopts alternative conformations when bound to targets that promote interference or priming in vivo. In addition, we identified Cse1 L1 loop mutations that switch Cascade to the priming conformation, changing the functional outcome of Cascade-target binding from interference to priming even when bound to interference targets. Our results demonstrate that Cascade conformation controls CRISPR immune response following target binding

Asymmetric thymocyte death underlies the CD4:CD8 T-cell ratio in the adaptive immune system

It has long been recognized that the T-cell compartment has more CD4 helper than CD8 cytotoxic T cells, and this is most evident looking at T-cell development in the thymus. However, it remains unknown how thymocyte development so favors CD4 lineage development. To identify the basis of this asymmetry, we analyzed development of synchronized cohorts of thymocytes in vivo and estimated rates of thymocyte death and differentiation throughout development, inferring lineage-specific efficiencies of selection. Our analysis suggested that roughly equal numbers of cells of each lineage enter selection and found that, overall, a remarkable ∼75% of cells that start selection fail to complete the process. Importantly it revealed that class I-restricted thymocytes are specifically susceptible to apoptosis at the earliest stage of selection. The importance of differential apoptosis was confirmed by placing thymocytes under apoptotic stress, resulting in preferential death of class I-restricted thymocytes. Thus, asymmetric death during selection is the key determinant of the CD4:CD8 ratio in which T cells are generated by thymopoiesis

Association between Plasma Antibody Response and Protection in Rainbow Trout Oncorhynchus mykiss Immersion Vaccinated against Yersinia ruckeri

A key hallmark of the vertebrate adaptive immune system is the generation of antigen-specific antibodies from B cells. Fish are the most primitive gnathostomes (jawed vertebrates) possessing an adaptive immune system. Vaccination of rainbow trout against enteric redmouth disease (ERM) by immersion in Yersinia ruckeri bacterin confers a high degree of protection to the fish. The immune mechanisms responsible for protection may comprise both cellular and humoral elements but the role of specific immunoglobulins in this system has been questioned and not previously described. The present study demonstrates significant increase in plasma antibody titers following immersion vaccination and significantly reduced mortality during Y. ruckeri challenge

A Selective Advantage for Conservative Viruses

In this letter we study the full semi-conservative treatment of a model for the co-evolution of a virus and an adaptive immune system. Regions of viability are calculated for both conservatively and semi-conservatively replicating viruses interacting with a realistic semi-conservatively replicating immune system. The conservative virus is found to have a selective advantage in the form of an ability to survive in regions with a wider range of mutation rates than its semi-conservative counterpart. This may help explain the existence of a rich range of viruses with conservatively replicating genomes, a trait which is found nowhere else in nature.Comment: 4 pages, 2 figure

Viral evolution under the pressure of an adaptive immune system - optimal mutation rates for viral escape

Based on a recent model of evolving viruses competing with an adapting immune system [1], we study the conditions under which a viral quasispecies can maximize its growth rate. The range of mutation rates that allows viruses to thrive is limited from above due to genomic information deterioration, and from below by insufficient sequence diversity, which leads to a quick eradication of the virus by the immune system. The mutation rate that optimally balances these two requirements depends to first order on the ratio of the inverse of the virus' growth rate and the time the immune system needs to develop a specific answer to an antigen. We find that a virus is most viable if it generates exactly one mutation within the time it takes for the immune system to adapt to a new viral epitope. Experimental viral mutation rates, in particular for HIV (human immunodeficiency virus), seem to suggest that many viruses have achieved their optimal mutation rate. [1] C.Kamp and S. Bornholdt, Phys. Rev. Lett., 88, 068104 (2002)Comment: 5 pages RevTeX including 3 figure