Selection of the lamprey VLRC antigen receptor repertoire

The alternative adaptive immune system of jawless vertebrates is based on different isotypes of variable lymphocyte receptors (VLRs) that are composed of leucine-rich repeats (LRRs) and expressed by distinct B- and T-like lymphocyte lineages. VLRB is expressed by B-like cells, whereas VLRA and VLRC are expressed by two T-like lineages that develop in the thymoid, a thymus-like structure in lamprey larvae. In each case, stepwise combinatorial insertions of different types of short donor LRR cassettes into incomplete germ-line genes are required to generate functional VLR gene assemblies. It is unknown, however, whether the diverse repertoires of VLRs that are expressed by peripheral blood lymphocytes are shaped by selection after their assembly. Here, we identify signatures of selection in the peripheral repertoire of VLRC antigen receptors that are clonally expressed by one of the T-like cell types in lampreys. Selection strongly favors VLRC molecules containing four internal variable leucine-rich repeat (LRRV) modules, although VLRC assemblies encoding five internal modules are initially equally frequent. In addition to the length selection, VLRC molecules in VLRC+ peripheral lymphocytes exhibit a distinct pattern of high entropy sites in the N-terminal LRR1 module, which is inserted next to the germ-line–encoded LRRNT module. This is evident in comparisons to VLRC gene assemblies found in the thymoid and to VLRC gene assemblies found in some VLRA+ cells. Our findings are the first indication to our knowledge that selection operates on a VLR repertoire and provide a framework to establish the mechanism by which this selection occurs during development of the VLRC+ lymphocyte lineage

Evolution of thymopoietic microenvironments

In vertebrates, the development of lymphocytes from undifferentiated haematopoietic precursors takes place in so-called primary lymphoid organs, such as the thymus. Therein, lymphocytes undergo a complex differentiation and selection process that culminates in the generation of a pool of mature T cells that collectively express a self-tolerant repertoire of somatically diversified antigen receptors. Throughout this entire process, the microenvironment of the thymus in large parts dictates the sequence and outcome of the lymphopoietic activity. In vertebrates, direct genetic evidence in some species and circumstantial evidence in others suggest that the formation of a functional thymic microenvironment is controlled by members of the Foxn1/4 family of transcription factors. In teleost fishes, both Foxn1 and Foxn4 contribute to thymopoietic activity, whereas Foxn1 is both necessary and sufficient in the mammalian thymus. The evolutionary history of Foxn1/4 genes suggests that an ancient Foxn4 gene lineage gave rise to the Foxn1 genes in early vertebrates, raising the question of the thymopoietic capacity of the ancestor common to all vertebrates. Recent attempts to reconstruct the early events in the evolution of thymopoietic tissues by replacement of the mouse Foxn1 gene by Foxn1-like genes isolated from various chordate species suggest a plausible scenario. It appears that the primordial thymus was a bi-potent lymphoid organ, supporting both B cell and T cell development; however, during the course of vertebrate, evolution B cell development was gradually diminished converting the thymus into a site specialized in T cell development

College completion predicts lower depression but higher metabolic syndrome among disadvantaged minorities in young adulthood

College graduates enjoy healthier, longer lives compared with individuals who do not graduate from college. However, the health benefit of educational attainment is not as great for blacks as it is for whites. Moreover, college completion may not erase the detrimental effects of early-life disadvantage for blacks and Hispanics. We use nationally representative data on young adults to test whether American minorities experience differential returns to educational attainment. We find that college completion predicts lower rates of depression for all racial groups. It also predicts lower metabolic syndrome among whites. However, college completion predicts higher metabolic syndrome among black and Hispanic adults from disadvantaged backgrounds, suggesting upward mobility may come at a health cost to young minorities in America

Evolutionary transition from degenerate to nonredundant cytokine signaling networks supporting intrathymic T cell development

In mammals, T cell development critically depends on the IL-7 cytokine signaling pathway. Here we describe the identification of the zebrafish ortholog of mammalian IL-7 based on chromosomal localization, deduced protein sequence, and expression patterns. To examine the biological role of il7 in teleosts, we generated an il7 allele lacking most of its coding exons using CRISPR/Cas9-based mutagenesis. il7-deficient animals are viable and exhibit no obvious signs of immune disorder. With respect to intrathymic T cell development, il7 deficiency is associated with only a mild reduction of thymocyte numbers, contrasting with a more pronounced impairment of T cell development in il7r-deficient fish. Genetic interaction studies between il7 and il7r mutants, and il7 and crlf2(tslpr) mutants suggest the contribution of additional, as-yet unidentified cytokines to intrathymic T cell development. Such activities were also ascertained for other cytokines, such as il2 and il15, collectively indicating that in contrast to the situation in mammals, T cell development in the thymus of teleosts is driven by a degenerate multicomponent network of γc cytokines; this explains why deficiencies of single components have little detrimental effect. In contrast, the dependence on a single cytokine in the mammalian thymus has catastrophic consequences in cases of congenital deficiencies in genes affecting the IL-7 signaling pathway. We speculate that the transition from a degenerate to a nonredundant cytokine network supporting intrathymic T cell development emerged as a consequence of repurposing evolutionarily ancient constitutive cytokine pathways for regulatory functions in the mammalian peripheral immune system

Antigen receptor repertoires of one of the smallest known vertebrates

The rules underlying the structure of antigen receptor repertoires are not yet fully defined, despite their enormous importance for the understanding of adaptive immunity. With current technology, the large antigen receptor repertoires of mice and humans cannot be comprehensively studied. To circumvent the problems associated with incomplete sampling, we have studied the immunogenetic features of one of the smallest known vertebrates, the cyprinid fish Paedocypris sp. “Singkep” (“minifish”). Despite its small size, minifish has the key genetic facilities characterizing the principal vertebrate lymphocyte lineages. As described for mammals, the frequency distributions of immunoglobulin and T cell receptor clonotypes exhibit the features of fractal systems, demonstrating that self-similarity is a fundamental property of antigen receptor repertoires of vertebrates, irrespective of body size. Hence, minifish achieve immunocompetence via a few thousand lymphocytes organized in robust scale-free networks, thereby ensuring immune reactivity even when cells are lost or clone sizes fluctuate during immune responses

Targeting TRAF6 E3 ligase activity with a small-molecule inhibitor combats autoimmunity

Constitutive NF-B signaling represents a hallmark of chronic inflammation and autoimmune diseases. The E3 ligase TNF receptor-associated factor 6 (TRAF6) acts as a key regulator bridging innate immunity, pro-inflammatory cytokines, and antigen receptors to the canonical NF-B pathway. Structural analysis and point mutations have unraveled the essential role of TRAF6 binding to the E2-conjugating enzyme ubiquitin-conjugating enzyme E2 N (Ubc13 or UBE2N) to generate Lys63-linked ubiquitin chains for inflammatory and immune signal propagation. Genetic mutations disrupting TRAF6 -Ubc13 binding have been shown to reduce TRAF6 activity and, consequently, NF-B activation. However, to date, no small-molecule modulator is available to inhibit the TRAF6 -Ubc13 interaction and thereby counteract NF-B signaling and associated diseases. Here, using a high-throughput small-molecule screening approach, we discovered an inhibitor of the TRAF6 -Ubc13 interaction that reduces TRAF6 -Ubc13 activity both in vitro and in cells. We found that this compound, C25-140, impedes NF-B activation in various immune and inflammatory signaling pathways also in primary human and murine cells. Importantly, C25-140 ameliorated inflammation and improved disease outcomes of autoimmune psoriasis and rheumatoid arthritis in preclinical in vivo mouse models. Hence, the first-in-class TRAF6 -Ubc13 inhibitor C25-140 expands the toolbox for studying the impact of the ubiquitin system on immune signaling and underscores the importance of TRAF6 E3 ligase activity in psoriasis and rheumatoid arthritis. We propose that inhibition of TRAF6 activity by small molecules represents a promising novel strategy for targeting autoimmune and chronic inflammatory diseases

Non-Invasive Detection of a Small Number of Bioluminescent Cancer Cells In Vivo

Early detection of tumors can significantly improve the outcome of tumor treatment. One of the most frequently asked questions in cancer imaging is how many cells can be detected non-invasively in a live animal. Although many factors limit such detection, increasing the light emission from cells is one of the most effective ways of overcoming these limitations. Here, we describe development and utilization of a lentiviral vector containing enhanced firefly luciferase (luc2) gene. The resulting single cell clones of the mouse mammary gland tumor (4T1-luc2) showed stable light emission in the range of 10,000 photons/sec/cell. In some cases individual 4T1-luc2 cells inserted under the skin of a nu/nu mouse could be detected non-invasively using a cooled CCD camera in some cases. In addition, we showed that only few cells are needed to develop tumors in these mice and tumor progression can be monitored right after the cells are implanted. Significantly higher luciferase activity in these cells allowed us to detect micrometastases in both, syngeneic Balb/c and nu/nu mice

Zebrafish Kidney Phagocytes Utilize Macropinocytosis and Ca2+-Dependent Endocytic Mechanisms

Background: The innate immune response constitutes the first line of defense against invading pathogens and consists of a variety of immune defense mechanisms including active endocytosis by macrophages and granulocytes. Endocytosis can be used as a reliable measure of selective and non-selective mechanisms of antigen uptake in the early phase of an immune response. Numerous assays have been developed to measure this response in a variety of mammalian and fish species. The small size of the zebrafish has prevented the large-scale collection of monocytes/macrophages and granulocytes for these endocytic assays. Methodology/Principal Findings: Pooled zebrafish kidney hematopoietic tissues were used as a source of phagocytic cells for flow-cytometry based endocytic assays. FITC-Dextran, Lucifer Yellow and FITC-Edwardsiella ictaluri were used to evaluate selective and non-selective mechanisms of uptake in zebrafish phagocytes. Conclusions/Significance: Zebrafish kidney phagocytes characterized as monocytes/macrophages, neutrophils and lymphocytes utilize macropinocytosis and Ca 2+-dependant endocytosis mechanisms of antigen uptake. These cells do not appear to utilize a mannose receptor. Heat-killed Edwardsiella ictaluri induces cytoskeletal interactions for internalization in zebrafish kidney monocytes/macrophages and granulocytes. The proposed method is easy to implement and should prove especially useful in immunological, toxicological and epidemiological research