Lamprey Variable Lymphocyte Receptors Mediate Complement-dependent Cytotoxicity

Lamprey, the most primitive jawless vertebrate, has a unique adaptive immunesystem consisting of variable lymphocyte receptors (VLRs) instead of IgG/IgM, TCRand BCR of jawed vertebrates.The lamprey lectin and alternative complement activation pathways have beendescribed, owing to the discovery of key complement components such as MBL,MASP, Bf, C3and other complement molecules, whereas the classicalimmunoglobulin-mediated complement activation pathway is absent in lamprey dueto the lack of IgG/IgM. Whether adaptive immune molecule VLRB participatingcomplement activation is not reported yet.We substantiated that the lamprey antigen-stimulated sera had obviously cytotoxiceffects on corresponding antigens, including RRBC, SRBC, the Gram-negativebacteria and tumor cells (NB4and Hela). However, the antisera had no cytotoxiceffects to Gram-positive bacteria, despite the fact that VLRB agglutination responsesto these bacteria were of the same magnitude as those observed againstGram-negative bacteria. This discrepancy may due to the fact that the Gram-positivecell wall is made of a dense layer typically composed of numerous rows ofpeptidoglycan and molecules of lipoteichoic acid, wall teichoic acid and surfaceproteins, which prevent them from complement attack. Normal as well as salinestimulated lamprey sera showed slight cytolytic effects on target cells, which may dueto the complement activations.To characterize cytotoxicity of the lamprey stimulated sera, RRBCs and E. coliwere incubated with respective antisera. The cytotoxicity of the stimulated sera wasdose-dependent. At a sera concentration of4%, about80%cells were lysed and thehigher concentrations (up to40%) had no further effects. We further demonstratedthat the cytotoxicity of antisera was temperature sensitive. The optimum temperature was between4°C and25°C, at which about80%cells were lysed. The cytotoxic effectdeclined when the temperature was above50°C and the antisera completely lostcytolytic effect when the temperatures reached56°C. In addition, the cytotoxicity ofantisera was time-dependent. The optimum time was20min and prolongedincubation (up to120min) did not increase the cytotoxicity. To determine whetherCa2+and Mg2+were required for the cytotoxic effects of the antisera, the E. colistimulated sera were incubated with serial dilutions of EGTA or EDTA. Removal ofCa2+and Mg2+from the stimulated sera by addition of EDTA or EGTA completelyabolished the bacteriolytic activity. Furthermore, the chelated antisera regained itsbacteriolytic activity by addition of excess Ca2+and Mg2+.More importantly, the cytolytic effects of lamprey antisera required VLRB,C1q-like and C3protein. The depletion of VLRB, C1q-like or C3protein resulted in asignificant reduction of the cytolytic activity.Solving the issue of how the complement system is activated by VLRB willrequire to elucidate the molecular mechanisms involving in this process. C1q hasrecently been discovered that it plays various roles in different complement activationpathways. As we know, C1q can activate the classical complement activation pathwayby binding the Fc portion of antigen-bound IgG/IgM in the higher vertebrates. Inaddition, SIGN-R1, a lectin that captures microbial polysaccharides, could directlybind C1q and assemble a C3convertase without the traditional requirement for eitherantibody or factor B. In lamprey, C1q-like molecule also performs dual function. Onone hand, acting as a GlcNAc-specific lectin, C1q-like associates with a serineprotease of the MASP/C1r/C1s family to activate C3in the lectin pathway. On theother hand, we found that C1q-like could also bind specific VLR. Their interactioncould make them deposit on the surface of exogenous cells antigens, lead tocomplement activation and finally cause target cells lysis.We also analyzed the expression and tissue distribution of C1q-like mRNA inlamprey. The C1q-like gene had the highest expression level in the intestine, followedby the heart, gill and kidney. No C1q-like gene was detected in the leucocytes or liver.Interestingly, Cooper research group reported that the antigen-binding VLRB cells distributed in the typhlosole, gill region and kidneys in lamprey. The similardistribution of C1q-like and VLR indicates that these tissues may be the primarybattlefront, in which the lectin pathway (the innate immunity) mediated byC1q-MASP-A may play a crucial role in the first line of defense by activating C3toclear the invasive pathogens. When the animals encounter the same antigens again,the immunological memory begins to function (the adaptive immunity). Theinteraction of antigen-specific VLRB and C1q-like molecule initiates the complementactivation.In summary, our data identified that an antigen-specific VLR-mediated andcomplement-dependent cytotoxicity in lamprey. The orchestration of VLR andcomplement system provides an important safeguard against invading microorganismand tumor cells. The powerful defense weapons might play a role in protectinglamprey, though the immunoglobulin and complement classical activation pathwayhave not yet emerged in it.