Systemic C3Modulates CD8~+T Cell Contraction And Memory Generation After Listeria Monocytogenes Infection

After intracellular bacterial and viral infections, antigen-specific CD8+T cells receiveantigen signal, constimulatory signal and cytokine signal from antigen presenting cells；CD8+T cells proliferate and differentiate into effector cells, and its cell numbers expandenormously to reach a peak at7-8day postinfection. As follows, the majority of effectorCD8+T cells undergo apoptosis while about10%survive and slowly turn into long-livedmemory T cels. According to the characteristic of dynamic curve of CD8+T cell response, itcould be divided into three phases: effector phase, contraction phase and memorymaintenance phase. During effector phase, the CD8+T cell proliferation is accompanied bydifferentiation into effector CD8+T cells that leave the secondary lymphoid organs to clearthe pathogen in site of infection. The contraction phase and memory maintenance phase arevery important, because the organism could avoid immunopathology due to excessiveimmune response and maintain flexibility to respond against new pathogens, and T cellmemory can provide stronger protective immune reponse to subsequent challenges by thesame pathogen. The mechanisms underline the regulation of CD8+T cell contraction andmemory generation are of vital importance. Complement system is one important part ofthe innate immunity to defend against foreign pathogens, and it is reported to have diverseroles in physiology, including regulating adaptive immunity. Complements can regulate Bcell immune in many stages and in many levels, and also regulate T cell differentiation,proliferation and apoptosis. There are many reports finding that complement can regulateprimary CD8+T cell response in many transplant and viral infection models, but nowwhether complement could regulate CD8+T cell contraction and memory generation are notclear.We used an intracellular pathogen, attenuated recombinant Listeria monocytogenesexpressing OVA (ΔrLm-OVA)to infect C3deficient mice (C3-/-), and observe the dynamiccurve of antigen-specific CD8+T cell response by intracellular staining of IFN-γ and Kb/OVA pentamer staining. The results showed that although primary CD8+T cell responsewas lower in C3-/-mice than in wild type (WT) mice, CD8+T cell contraction was reducedand memory generation was increased in C3-/-mice. Using the recombinant Listeriamonocytogenes expressing OVA (rLm-OVA), we also found reduced CD8+T cellcontraction and increased memory generation in C3-/-mice compared to WT mice.Cobra Venom Factor (CVF) could be used to treat mice and deplete the circulating orsystemic complement component C3. We tried to use CVF to treat mice (-1to14day) andfound that the systemic C3of treated mice reduced to about1/10of its normal level andsustained about one week. Reduced CD8+T cell contraction and increased memorygeneration were found in C3depleted mice treated with CVF as compared to control mice.Thus we suggest that systemic complement play an important role in regulating CD8+T cellresponse.A lot of articles find that local production by APC and T cells could play an importantrole in regulating T cell immune. To further clarify the roles of systemic complementcomponent from hepatic source or local produced C3by monocytes and lymphocytes inregulating CD8+T cell response, we made bone marrow (BM) chimeric mice to do theresearch. BM chimeric mice have systemic C3from the liver of recipient mice and local C3produced by monocytes and lymphocytes in second lymphoid organ from donor BM cells.This chimeric mice have the advantage of distinguish between the role of systemic or localC3in regulating CD8+T cell contraction and memory generation. C3-/-and WT recipientmice were irradiated by sublethal dose of γ radiation and transferred with cogenic BM cellsfrom C3-/-and WT donor. There were four groups of BM chimeric mice. After two month,these BM chimeric mice were infected with ΔrLm-OVA, and the changes in CD8+T cellresponses were analyzed using peripheral blood lymphocytes (PBMCs) postinfection. Wefound that the C3-/-recipients exhibited slower contraction compared with WT recipientswhen engrafted with BM cells from the same donor. In contrast, CD8+T cell contraction inthe same recipient mice when engrafted with BM cells from C3-/-or WT donors,respectively, showed no significant difference. These data suggest that systemic C3play animportant role in regulating CD8+T cell contraction and memory generation.The result that systemic C3depleted by CVF can modulate CD8+T cell contraction andmemory generation indicate that it could be apply to improving the efficacy of vaccine design. We concerned about whether memory cells generated in C3deficient or depletedmice have normal function. Memory CD8+T cells have unique phenotype and functiondiffident from effector CD8+T cells and naive CD8+T cells. First, we first analyzed thephenotype of memory CD8+T cells between C3-/-and WT mice. We found that theexpression of CD62L, KLRG-1, CD127, CD44and the secretion of IL-2in C3-/-mice didnot have significant statistical difference. Second, differently labeled target cells loaded orunloaded with OVA peptide were transferred to C3-/-and WT mice during memorymaintenance phase, and the in vivo killing assay were performed to determine the cytotoxicability of memory CD8+T cells. The memory CD8+T cells in C3-/-mice killedapproximately two fold OVA-specific target cells compared with those in WT mice. Sincethe amount of memory T cells in C3-/-mice was about twice of that in WT mice, theseresults indicate that the cytotoxic potential of memory cells in C3-/-mice was comparable tothat in WT mice. Third, when CVF treated mice and control mice were given secondaryinfection, the CD8+T cell response in both group had comparable magnitude of expansion.This result indicates that memory CD8+T cells generated in C3depleted mice have the samesecondary response ability as in control mice. Last, we isolated memory CD8+T cells fromC3-/-and WT mice and transferred these cells into naive recipient mice. Then the recipientmice were challenged with ΔrLm-OVA, and the Ag-specific CD8+T cell response weredetermined. We found that memory CD8+T cells transferred from C3-/-and WT mice hadsimilar fold of expansion after challenge. These results show that memory CD8+T cellsgenerated from C3deficient or depleted mice have similar phenotype and function as thosein WT mice.CD8+T cell response could be regulated by many distinct factors during effector orcontraction phase. We tried to use CVF to treat mice during effetor phase(-1to6day) andcontraction phase(7to14day) to ascertain during which phase C3regulate the CD8+T cellcontraction and memory generation. We found that in mice depleted C3during effectorphase CD8+T cell contraction were reduced than that in control mice, but in mice depletedC3during contraction phase CD8+T cell contraction were comparable to that in controlmice. These data show that C3play a role in regulating CD8+T cell contraction and memorygeneration during effector phase. We then focused to analyze whether those factors knownto program CD8+T cell response during effector phase were involved in the regulation of CD8+T cell contraction and memory generation by C3.To start with, some literature reported that antigen persistence during late effetor phasecould impact CD8+T cell differentiation and contraction. We treated C3-/-and WT micewith ampicillin from day4for a week after infection to eliminate persistent bacteria. CFUexperiment confirmed that the bacteria were eliminated from C3-/-and WT mice during lateeffector phase. OVA-specific CD8+T cells in C3-/-mice contracted much slower andresulted in more OVA-specific memory CD8+T cells as compared to WT mice. Theseresults exclude the possibility that the reduced CD8+T cell contraction is due to theshortened duration of Ag persistence. Moreover, Ag-specific effector CD8+T cells is apopulation of heterogeneity composed by many T cell colones of different avidity. Thecolones of high avidity are much easier to differentiate to short-lived effector cells than thatof low avidity. We then attempted to determine the avidity of effector CD8+T cells at thepeak response in C3-/-and WT mice. We found that the Vβ usage of the effector CD8+Tcells between the two group have no significant difference. Moreover, effector CD8+T cellsin C3-/-mice have similar sensitivity in response to different titer of specific peptide. So theavidity of effector CD8+T cells is not associated with different contraction between the C3-/-and WT mice. All these results suggested that antigen related factors might not correlatewith the process of C3regulating CD8+T cell contraction and memory generation.Later, we found that KLRG-1and CD127expression among effetor CD8+T cellsbetween C3-/-and WT mice were significantly different. In C3-/-mice fewer effector CD8+Tcells differentiated into KLRG-1hiCD127lowshort-lived effector cells (SLECs) and moreinto KLRG-1lowCD127hilong-lived memory precursor effector cells (MPECs). KLRG-1hieffector subsets in CVF treated mice were fewer than control mice at the peak of theresponse. These results indicate that different differentiation of effector CD8+T cells intoSLECs or MPECs correlate with the significantly different contraction and memorygeneration between C3deficient (or depleted) mice and control mice. Moreover, there aremany literature support that inflammatory cytokines play a very import role inprogramming CD8+T cell differentiation, contraction and memory generation. We then triedto determine whether the levels of inflammatory cytokines were different between C3-/-andWT mice at24h post infection, and we found that the levels of IL-12, IFN-γ, etc.expression were significantly reduced in C3-/-mice compared to WT mice. Together with previous results, one possible mechanism that could explain how C3regulate CD8+T cellresponse is that reduced inflammatory cytokines in C3-/-mice program the CD8+T celldifferentiation and result in reduced CD8+T cell contraction and increased memorygeneration.In addition, many research found that local C3produced by APCs and T cell mediatethe regulation of T cell immune through C5aR signaling pathway. We first adopted C5aRantagonist to inhibit the C5aR signaling pathway and determined how it affects the dynamiccurve of Ag-specific CD8+T cell response. We found that C5aR antagonist treated groupshowed no apparent difference in CD8+T cell contraction and memory generation ascompared to control group. Further, we used C5aR deficient mice to determine whetherC5aR signaling was involved in the regulation of CD8+T cell contraction, and found thatthe CD8+T cell contraction and memory generation had no difference between C5aRdeficient and WT mice. These results suggest that C3could regulate CD8+T cell contractionand memory generation not depending on C5aR signaling pathway which is downstream ofC3activation.Our results reveal an important role for systemic C3in influencing CD8+T cellcontraction, which add up to our knowledge that the mechanisms by which complementregulates the CD8+T cell response, and provide insights into the interaction of innateimmune and adaptive immune. Further-more, because depletion of C3by CVF treatmentreduced CD8+T cell contraction and increased the generation of CD8+memory T cells withenhanced protective function, these findings suggest that strategies that alter the functionsof the complement pathway may improve strategy of vaccine design and enhance vaccineefficacy.