Alternative Adaptive Immune System Research Articles

Evolution of two distinct variable lymphocyte receptors in lampreys: VLRD and VLRE

Jawless vertebrates possess an alternative adaptive immune system in which antigens are recognized by variable lymphocyte receptors (VLRs) generated by combinatorial assembly of leucine-rich repeat (LRR) cassettes. Three types of receptors, VLRA, VLRB, and VLRC, have been previously identified. VLRA- and VLRC-expressing cells are Tcell-like, whereas VLRB-expressing cells are B cell-like. Here, we report two types of VLRs in lampreys, VLRD and VLRE, phylogenetically related to VLRA and VLRC. The germline VLRD and VLRE genes are flanked by 39 LRR cassettes used in the assembly of mature VLRD and VLRE, with cassettes from chromosomes containing the VLRA and VLRC genes also contributing to VLRD and VLRE assemblies. VLRD and VLRE transcription is highest in the triple-negative (VLRA-/VLRB-/VLRC-) population of lymphocytes, albeit also detectable in VLRA+ and VLRC+ populations. Tissue distribution studies suggest that lamprey VLRD+ and VLRE+ lymphocytes comprise T-like sublineages of cells.

An inhibitory receptor of VLRB in the agnathan lamprey.

Lamprey, the primitive jawless vertebrate, uses variable lymphocyte receptor (VLR) as alternative adaptive immune system instead of immunoglobulin (Ig)-based receptors used in jawed vertebrates. In the present study, we characterized a potential inhibitory receptor of VLRB from leucocytes in lamprey. It is a novel ITIM-containing IgSF protein and was therefore named as NICIP. NICIP has two Ig-like domains in extracellular region, a transmembrane domain and two classical ITIM motifs in cytoplasmic domain. It is mainly expressed on the surface of granulocytes and monocytes and can interact with VLRB. In transiently transfected HEK293T cells, it was confirmed again that it could interact with VLRB and the two phosphorylated ITIM motifs could recruit SHP-1 and SHP-2. These results imply that NICIP may play a role as a potential inhibitory receptor of VLRB and involve in negative regulation of immune response mediated by VLRB.

Investigation of variable lymphocyte receptors in the alternative adaptive immune response of hagfish

Jawless vertebrates have an alternative adaptive immune system mediated by variable lymphocyte receptors (VLRs), VLRA, VLRC and VLRB. In investigation on the adaptive immunity of hagfish, avian influenza virus hemagglutinin (H9N2-HA1) was used as a model antigen, with mRNA expression levels of VLRA, VLRC and Ikaros were up-regulated in the first week post-immunization. CD45 was up-regulated after the first week; and expression of VLRB progressively increased over the course of the trial. The transcriptional/translational activation of VLRB in blood was verified. The VLRBs cloned from these transcripts showed diversity in their leucine-rich repeats (LRRs). The production of specific VLRB increased in a time- and dose-dependent manner, detected by an anti-VLRB antibody (11G5). The plasma VLRB could distinguish H9N2-HA1 from unrelated proteins, but not from other HA1 subtypes. Together, our findings show that VLRs play a major role in the alternative adaptive immune system of hagfish by responding to specific foreign substances, such as H9N2-HA1.

EVENTS

Jawless vertebrates have an alternative adaptive immune system mediated by variable lymphocyte receptors (VLRs), VLRA, VLRC and VLRB. In investigation on the adaptive immunity of hagfish, avian influenza virus hemagglutinin (H9N2-HA1) was used as a model antigen, with mRNA expression levels of VLRA, VLRC and Ikaros were up-regulated in the first week post-immunization. CD45 was up-regulated after the first week; and expression of VLRB progressively increased over the course of the trial. The transcriptional/translational activation of VLRB in blood was verified. The VLRBs cloned from these transcripts showed diversity in their leucine-rich repeats (LRRs). The production of specific VLRB increased in a time- and dose-dependent manner, detected by an anti-VLRB antibody (11G5). The plasma VLRB could distinguish H9N2-HA1 from unrelated proteins, but not from other HA1 subtypes. Together, our findings show that VLRs play a major role in the alternative adaptive immune system of hagfish by responding to specific foreign substances, such as H9N2-HA1.

EVENTS

Jawless vertebrates have an alternative adaptive immune system mediated by variable lymphocyte receptors (VLRs), VLRA, VLRC and VLRB. In investigation on the adaptive immunity of hagfish, avian influenza virus hemagglutinin (H9N2-HA1) was used as a model antigen, with mRNA expression levels of VLRA, VLRC and Ikaros were up-regulated in the first week post-immunization. CD45 was up-regulated after the first week; and expression of VLRB progressively increased over the course of the trial. The transcriptional/translational activation of VLRB in blood was verified. The VLRBs cloned from these transcripts showed diversity in their leucine-rich repeats (LRRs). The production of specific VLRB increased in a time- and dose-dependent manner, detected by an anti-VLRB antibody (11G5). The plasma VLRB could distinguish H9N2-HA1 from unrelated proteins, but not from other HA1 subtypes. Together, our findings show that VLRs play a major role in the alternative adaptive immune system of hagfish by responding to specific foreign substances, such as H9N2-HA1.

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.

Cellular and molecular characterization of hagfish VLR-based adaptive immune system (VET2P.1043)

Abstract Jawless vertebrates have an alternative adaptive immune system in which variable lymphocyte receptors (VLR) are somatically diversified through recombinatorial assembly of leucine-rich repeat cassettes during lymphocyte development. Three VLR loci (VLRA, VLRB, and VLRC) have been defined in lampreys and each is expressed by a distinct lymphocyte population. Lamprey VLRA and VLRC are expressed by T-like lineages of lymphocytes, whereas VLRB is expressed by a B-like lineage of lymphocytes, much like αβ T, γδ T and B cells in jawed vertebrates. Recently we have revised the nomenclature for the hagfish VLRs by defining a third VLR gene. Here, monoclonal and polyclonal antibodies were generated against invariant residues of hagfish VLRA, VLRB and VLRC and these were used to identify VLR-bearing lymphocytes and their products. We found that hagfish VLRA, VLRB and VLRC are expressed by three separate lymphocyte populations. Size-exclusion chromatography and western blot analysis indicated that multivalent VLRB proteins are released into the circulation, which suggests that hagfish VLRB+ cells are B-cell like. In contrast, neither VLRA nor VLRC was detectable in the plasma, supporting the hypothesis that these two lymphocyte populations are T-cell like. The demonstration of three lymphocyte populations in both lampreys and hagfish suggests that this tripartite lymphocytic differentiation program already existed in a common ancestor of the two cyclostome lineages around 480 Mya.

Shrimp Immune System-Special Focus on Penaeidin

In this review paper, we summarize the immune system of penaeid shrimp. This review includes the clotting system comprising clot protein and clotting enzyme (transglutaminase II), as well as the prophenoloxidase (proPO) activation system including proPO-activating enzyme (PPAE), proPO and clip domain serine protease homolog (c-SPH). In addition, several shrimp cytokines or cytokine-like molecules found in tiger shrimp Penaeus monodon participating in the defense responses are also discussed. C-SPH could enhance hemocyte adhesion. Shrimp astakine promoted hemocyte proliferation in the hematopoietic tissues and could be down-regulated at the post-transcriptional level by the binding of intracellular molecules such as transglutaminase I and crustin Pm4 to its 3'-untranslatd region. Penaeidin is found to be a dual function molecule as an antimicrobial peptide and an autocrine-acting cytokine. Yet, other antimicrobial substances such as quinone and melanine which are generated during the proPO activation pathway and the reactive oxygen and nitrogen intermediates (ROI and RNI) which are generated during the phagocytic process can also kill and clear the invading microorganisms directly. In addition, Dscam (Down syndrome cell adhesion molecule), a member of the immunoglobulin superfamily (IgSF) which plays an essential role in the alternative adaptive immune system of invertebrates, has also been characterized in shrimp.Penaeid shrimp is one of the most important commercially available aquaculture resources. Although, the culture techniques and field management skills have significantly improved in the recent years, infectious diseases are still a threat to the industry. The study of shrimp immune system could be important in designing the strategies against pathogen infection. For shrimp, the innate immune responses play a major role in combatting invading pathogens and prevent them against diseases. The immune responses involve different factors such as physical barrier, bacteria clearance, encapsulation, clotting reactions, prophenoloxidase system, reactive oxygen intermediates, and antimicrobial activity. The innate immune system uses a variety of pattern recognition receptors (PRR) on the hemocyte membrane to detect the pathogenassociated molecule pattern (PAMP) and transduce signals during pathogen invasion. Hemocytes are then activated and the defense molecules are degranulated against the pathogens.

Definition of a thirdVLRgene in hagfish

Jawless vertebrates (cyclostomes) have an alternative adaptive immune system in which lymphocytes somatically diversify their variable lymphocyte receptors (VLR) through recombinatorial use of leucine-rich repeat cassettes during VLR gene assembly. Three types of these anticipatory receptors in lampreys (VLRA, VLRB, and VLRC) are expressed by separate lymphocyte lineages. However, only two VLR genes (VLRA and VLRB) have been found in hagfish. Here we have identified a third hagfish VLR, which undergoes somatic assembly to generate sufficient diversity to encode a large repertoire of anticipatory receptors. Sequence analysis, structural comparison, and phylogenetic analysis indicate that the unique hagfish VLR is the counterpart of lamprey VLRA and the previously identified hagfish "VLRA" is the lamprey VLRC counterpart. The demonstration of three orthologous VLR genes in both lampreys and hagfish suggests that this anticipatory receptor system evolved in a common ancestor of the two cyclostome lineages around 480 Mya.

Immunoglobulin superfamily protein Dscam exhibited molecular diversity by alternative splicing in hemocytes of crustacean, Eriocheir sinensis

Be absent of adaptive immunity which have both specificity and memory, invertebrates seem to have evolved alternative adaptive immune strategies to resist various intruding pathogens. Whereas vertebrates could generate a wide range of immunological receptors with somatic rearrangement, invertebrates possibly depend on alternative splicing of pattern-recognition receptors (PRRs). Recently, it has been suggested that a member of the immunoglobulin superfamily (IgSF), Down syndrome cell adhesion molecule (Dscam), plays a crucial role in the alternative adaptive immune system of invertebrates. At present, we successfully isolated and characterized the first crab Dscam from Eriocheir sinensis. EsDscam has typical domain architecture compared with other Dscam orthologs, including one signal-peptide, 10 immunoglobulin (Ig) domains, 6 fibronectin type III domains (FNIII), one transmembrane domain (TM) and one cytoplasmic tail. We had detected four hypervariable regions of EsDscam in the N-terminal halves of Ig2 (25) and Ig3 domain (30), the complete Ig7 (18) and also the transmembrane domain (2), potentially generate 27,000 unique isoforms at least. Transcription of EsDscam were both a) detected in all tissues, especially in immune system, digestive system and nerve system; b) significantly induced in hemocytes post lipopolysaccharides (LPS), peptidoglycans (PG) and β-1, 3-glucans (Glu) injection. Importantly, we had detected membrane-bound and secreted Dscam isoforms in E. sinensis, and showed that secreted isoforms were extensively transcribed post different PAMPs challenge respectively. Results from immuno-localization assay revealed that EsDscam evenly distributed in the cell surface of hemocytes. These findings indicated that EsDscam is a hypervariable PRR in the innate immune system of the E. sinensis.

Lamprey variable lymphocyte receptors mediate complement-dependent cytotoxicity (172.20)

Abstract An alternative adaptive immune system is present in the most basal vertebrates, lampreys and hagfish, the only surviving jawless vertebrates. These eel-like fish use leucine-rich-repeat (LRR)-based receptors, called variable lymphocyte receptors (VLRs), for antigen recognition instead of the immunoglobulin (Ig)-based receptors used in jawed vertebrates. We showed that in Japanese lamprey (Lampetra japonica), antigen-specific VLRB antibodies interact with C1q-like and C3 proteins to mediate complement-dependent cytotoxicity for bacteria and tumor cells. The immune based lysis involved deposition of VLRB and C1q-like protein complex on the surface of target cells, activation of C3, and ultimate disruption of cell wall integrity. The demonstration of functional interaction between VLRB and complement components in lamprey provides evidence for the emergence of cooperative innate and adaptive immune responses at a pivotal point in vertebrate evolution, before or in parallel with the evolution of Ig-based antibodies and the classical complement activation pathway.

The putative invertebrate adaptive immune protein Litopenaeus vannamei Dscam (LvDscam) is the first reported Dscam to lack a transmembrane domain and cytoplasmic tail

It has recently been suggested that Dscam (Down syndrome cell adhesion molecule), a member of the immunoglobulin superfamily (IgSF), plays an essential role in the alternative adaptive immune system of invertebrates. Here, we isolated and characterized the first shrimp Dscam from Litopenaeus vannamei. The LvDscam protein had an extracellular domain but lacked the expected transmembrane domain and cytoplasmic tail, both of which are found in all other members of the Dscam family (and may also be found in other L. vannamei Dscams that have not yet been isolated). In nervous tissue, expression levels of LvDscam were unexpectedly low. Phylogenetic analysis suggests that LvDscam is far from the Dscams found in other invertebrates. Nevertheless, the domain architecture of the extracellular region of LvDscam is similar to other invertebrate Dscams, and it exhibits the typical configuration of 10 immunoglobulin (Ig) domains, 6 fibronectin type 3 domains (FNIII) and one cell attachment sequence (RGD). Cloning and characterization of a total of 62 cDNAs from hemocytes collected from WSSV-free, WSSV-persistent and WSSV-acute-infected shrimp revealed 23 alternative amino acid sequences in the N-terminal of Ig2, 30 in the N-terminal of Ig3 and 13 in the Ig7 domain. This implies that LvDscam can potentially encode at least 8970 unique isoforms. Further analysis suggested that the LvDscam Ig2 and Ig3 regions are more functionally important than Ig7 in the shrimp's specific immune response against WSSV. We discuss how this tail-less, soluble Dscam can still play an active role in alternative adaptive immune response even while its axonal guidance functionality may be impaired.