Origins Of Adaptive Immune System

When and how adaptive immunity emerged is one of the fundamental questions in immunology.Lymphocytes with a clonally diverse anticipatory repertoire are the key player for the adaptive immune system,each lymphocyte bearing a unique antigen receptor.Rearranging TCR and BCR receptor genes and the MHC,were previously considered to be a unique character to the lymphocytes and the adaptive immune system was emerged suddenly during the jawless-jawed vertebrate transition.Recent study of the surviving jawless vertebrates(lampreys and hagfish)revealed that the lymphocytes in these jawless vertebrates use alternative ways to solve the receptor diversification problem,namely by the recombinatorial assembly of LRR(leucine-rich-repeat)genetic modules to encode variable lymphocyte receptors in recognizing and repelling pathogen invaders.Accumulating data show the morphological clues of possible lymphocytes(referred to as lymphocyte-like cells)in protochordate ascidian and amphioxus with a possible clonally diverse anticipatory repertoire in protochordate amphioxus.Although these lymphocyte-like cells resemble morphologically the vertebrate lymphocyte,whether they are indeed evolutionarily related to lymphocytes or arose through parallel evolution remains unclear.These data have revealed that the transcription factors related to the formation and differentiation of lymphocytes are evolutionarily conserved.The study of the transcription factors will provide key evidence in determining whether these prochordate lymphocyte-like cells are indeed genetically related to lymphocytes or arose through parallel evolution.Cephalochordata represents a stem lineage of the chordates,which gave rise to both the urochordate and vertebrate clades.Amphioxus(or lancelet)is the living representative of the Cephalochordata.In this dissertation,the amphioxusBranchiostoma belcheri is used as mode animal for challenging the question of the origination of lymphocyte cells.All the samples came from the cultivation center of Beihai Marine,Nanjing University and their wild population lives in the shallow sea of the coastal area of Southern China Sea,Guangdong.Lymphocyte cells are morphologically distinct and conserved among the vertebrates and they are small,mostly rounded with a relatively large nucleus,which is spherical and densely compacted.The scanty cytoplasm lacks ribosomes and has scattered mitochondria.Intensive TEM observation of Branchiostoma belcheri has revealed the presence of lymphocyte-like cells,which reside densely among the branchial bars in an aggregated pattern.Additionally,we found that there are kinds of cells which are similar with lymphocyte-related cells of mammals,including plasma cells,effector T cells,mast cells,and so on.To examine whether the lymphocyte-like cells are genetically related to vertebrate lymphocytes and whether the gill bars are the site for lymphopoiesis,22 genes homologous to the vertebrate's transcription factors crucial for both hematopoiesis and lymphopoiesis were cloned from neurula samples or LPS-challenged samples and the whole amount in situ hybridization analyses of these transcription factors were performed.These transcript factors are:BbSpi-related,BbEtsl/2,Pax2/5/8,BbIkaros,Notch,c-myb,GATA1/2/3,hhex,runx and E2A.Among them,several transcription factors(GATA1/2/3,BbSpi-related,Bbikaros,c-myb,hhex and runx)are mainly related to hematopoiesis,some(BbSpi-related,GATA1/2/3,Bbikaros,BbEts1/2,c-myb and Notch)are related to myeloid differentiation,and most of them are related to development of lymphocytes,except c-myb and hhex.Some amphioxus transcription factors are the ancestors of their homologue or paralogue genes in vertebrates,and thus,they might bear the common function of the vertebrate genes.For example,vertebrate GATA1,GATA2 and GATA3 arise from their common ancestor GATA1/2/3 as a result of two whole genome duplication,and we suppose that amphioxus GATA1/2/3 bears the original function of GATA1,GATA2 and GATA3.The transcription factor BbSpi-related is a common ancestor of the vertebrate PU.1 family which consists of 4 members(Pu.l,Spi-B,Spi-C and Spi-D).PU.1 family's expression is mainly restricted to hemopoietic cells and tissues,playing a critical role in hemopoietic,myeloid and lymphocyte development.The whole mount in situ hybridization result of BbSpi-related shows that BbSpi-related expressed at the tailbud of neurula,then displayed in 2-anatomically separate patterns(the gill area and the tailbud)in the early larva stage,and interestingly,the caudal signal disappeared when larva was 2 weeks old.In addition to Pu.l,whole amount in situ hybridization of most transcription factors(including GATA1/2/3,Bbikaros,c-myb)related to hematopoiesis resembling that of zebrafish displays in two anatomically separate patterns in the early larval stage.The rostral expression area residing among the branchial bars and the caudal area among the tail bud.Among the mentioned transcription factors,Hhex is a key transcription factor playing a crucial role in hematopoietic differentiation.The expression of this gene in B.belcheri is restricted to the endostyle from neurula to larva,which indicates that amphioxus endostyle may not only be the homologous organ of the vertebrate thyroid,but plays an important role in the immune system.The Notch signaling affects differentiation of both myeloid cells and B/T lymphoid cells.The gene homologous to Notch-1 was isolated from amphioxus.The expression pattern of this gene is identical to that of BbSpi-related.Amphioxus c-Myb gene is conservative with its homologous genes in vertebrates,thus,it may play a similar role in mediating hematopoiesis and myeloid development as in vertebrates.The gene homologous to c-myb gene in B.belcheri was expressed with high levels restricted among the gill arches,and weaker signal in the gut.Paired box(Pax)genes family consists of four subfamilies,e.g.,Pax-1/9,Pax-3/7,Pax-4/6 and Pax-2/5/8.Pax-2/5/8 was subfunctioned into 3 genes,known as Pax-2,5 and 8 in vertebrates,but in amphioxus it is only represented by a single gene,known as AmphiPax2/5/8,which is located at the base of a divergent into separate Pax-2,5 and 8 genes in vertebrate evolution.The study of expression patterns indicates thatAmphiPax2/5/8 has a function shared separately with vertebrate Pax-2,5 and 8 genes.The transcription factor Pax-5 in vertebrates is expressed during most stages of B cell differentiation and influences the expression of several B cell-specific genes.The expression domain of AmphiPax2/5/8 precisely overlaps with other transcription factors for lymphopoiesis,which suggest that Pax-2/5/8 also plays a role in lymphocyte development similar to Pax-5 in vertebrates.In sum,the expression of these transcription factors through in situ hybridization gives us evidence for the origin of amphioxus hematopoiesis,and it is undoubted that there is indeed a complicated pathway of immune-related transcriptional factors in different developmental stages.According to the expression patterns,we propose a hypothesis that the gill area and tailbud might be the primitive hemopoietic organs of amphioxus,while the gill area,maybe including the endostyle,becomes a definitive hemopoietic organ when the larva is older.The expression profiles of the different transcription factors in different developmental stages were examined by real-time PCR,displaying that definitive hematopoiesis of amphioxus may occur at two weeks or sometime before.When amphioxus was challenged by LPS,Pax2/5/8 expression reduced while blimp-1,which is a key factor in restrictingplasma cells(effect B cells)increased.The result is strikingly similar to how vertebrates react when infected by exotic pathogens,which indicates that in the cephalochordata amphioxus,the original terminal differentiation of lymphocytes might appear.Lymphocytes have mostly a short lifespan and the primary lymphoid organs are unique sites for generating lymphocytes to maintain its population size.The PHA(T-cell mitogen)and LPS(regarded as B-cell mitogen)challenging in vertebrates induce a proliferative response in T-cells and B-cells,respectively.EdU(5-ethynyl-29-deoxyuridine)is widely applied as a marker for cell proliferation.Using the techniques mentioned,several parallel examinations were operated.The data reveal that the EdU in PHA challenging early larvae at the 3 gill bar stage was stained specifically in both the 2nd and 3rd-gill bars at a lower level at 6 hr but increasingly at higher levels in the whole gill area at 24 hr after challenging.The 1st gill bar both at 6 and 24 hr was weakly stained.EdU stain in wild cultured samples was however not concentrated in gill bars.The proliferation pattern of LPS challenging is similar to that of PHA challenging,but the quantity of proliferating cells is much less,with scattered signals around the 2nd and 3rd gill bars.The EdU evidence shows that lower proliferation patterns occured when LPS challenged than PHA did,which might reveal that LPS and PHA induce the proliferation of different types of cell-lineages.The production of large numbers of lymphocytes,especially during immune challenging,is balanced by their elimination via apoptosis.TUNEL is a common method for detecting DNA fragmentation resulting from apoptotic signaling cascades.TUNEL assay reveals that gill bars are the active site of apoptosis.The apoptosis occurred subsynchronically with the proliferation of the lymphocyte cells but slightly earlier after LPS challenging.Our data show that a noticeable apoptosis occurred already at 12 hrs after LPS challenging.The mast cells are one of key immune cells playing significant role both in innate and adaptive immune system and the reliable mast cells are reported from gnathan vertebrates only.Our project selects Branchiostoma belcheri as experimental animal to expose mast cells at basal chordate evolutionary history and to reveal their role in the rise of adaptive immune system.TEM observations and Histochemical Staining reveal presence of granular cells from Branchiostoma belcheri,which show striking resemblance to mammalian mast cell morphologically.The mast cell identity is also confirmed by immunehistochemical staining of histamine,heparin,serotonin and trypase),which demonstrate that the mast cell granules are filled with same mediators of mammalian mast cells.Degranulation of Mast cells occurred as response to parasite,bacteria and degranulating agent in natural and experimental conditions.Compound 48/80 originally described as a most active histamine-liberator agent.In mammalian,compound 48/80 is a potent degranulation agent of Mast cells,and in amphioxus,compound 48/80 presented to be a efficient activator of degranulation.Also,we found that amphioxus mast cell have a strong capacity of pathogen phagocytosis.Present study demonstrates presence of mast call back at basal chordates and suggests that the first mast cells being as potent phagocytosis and sentinels at immune frontier may have played as a trigger to the emergence of adaptive immune system.To date,little is known about how microRNAs(miRNAs)are involved in the immune defense in amphioxus.We identified the amphioxus miRNAs in the acute-phase response to Lipopolysaccharide(LPS)and chose 12 hr as the point to perform miRNA microarray analysis to select the differentially expressed miRNAs.Furthermore,we used quantitative real-time PCR to detect the expression patterns of selected amphioxus miRNAs under effective LPS challenge during the time course.The microarray data revealed that the miRNA expression file was significantly changed after LPS stimulation.The changes of the 10 most upregulated and 7 most downregualted miRNAs in gills of the amphioxus following challenge with LPS revealed a temporal induction kinetic.This study will provide valuable information to take an insight into molecular mechanism of innate immune and the evolution of the miRNA family.In addition,we focused on characterizing the miRNA expression patterns of three amphioxus digestive organs(the gill,intestine and hepatic caecum)that are believed to be associated with evolution of the vertebrate immune system.We identified the global miRNA expression profiles and discovered eight expression patterns in these organs.Meanwhile,we statistically compared the miRNA expression in paired organs and found that the expressions of specific miRNAs are significant difference in distinct organs.Furthermore,we validated the expression of 18 miRNAs in 6 different tissues.The validation analysis discovered that certain miRNAs showed tissue-specific expression pattern.For instance,bbe-miR-92c-3p is highly expressed in the gill but not in other 5 tissues,while bbe-miR-4869-5p is mainly expressed in the intestine.In addition,bbe-miR-4867-3p and bbe-miR-182b-5p specifically present in the hepatic caecum.This study thus fills in a knowledge gap by charactering miRNA expression indigestive organs that are associated with immune system evolution,and identifies tissue-specific miRNAs that may contribute to origin and evolution of the digestive system and immune system.In summary,all of the data and findings presented in the thesis provide clues that shed new light on the mystery of the evolutionary origin of the adaptive immune system.TEM observation and whole amount in situ hybridizations of the genes homologous to key transcription factors related to lymphopoiesis in vertebrates suggest that the lymphocyte-like cells in amphioxus likely are true lymphocytes,which are genetically related to lymphocytes in vertebrates,thus pushing the evolutionary origin of lymphocyte cells into the evolutionary roots of the Chordata.In addition,the PHA(T-cell mitogen)and LPS(be regarded as B-cell mitogen)challenging examination for proliferative response suggests that the lymphocyte cell lineages corresponding to vertebrate's T-cells and B-cells may have also evolved in the early chordates.Whole amount in situ hybridization of most transcription factors(including BbSpi-related,GATA1/2/3,Bbikaros,c-myb)related to hematopoiesis display in two anatomically separate patterns in the early larval stage,with these two sites being comparable to the transient site at the tail bud(with erythron fate).We propose a hypothesis that the gill area and tailbud might be the primitive hemopoietic organs of amphioxus,while the gill area(including the endostyle),become a definitive hemopoietic organ.