| Objective: Follicular helper T cell (Tfh cell) as a novel subset of CD4+helping T cell, Tfh cellplays very important roles in germinal center formation，B cell development, antibody classswitching and long term humoral immunity response establishment. While interferon-a (IFN-a), as an adjuvant to recombinant adenovirus vaccine can enhance effective immune responses.However, it is unclear whether IFN-a as an adjuvant plays the role in recombinant vaccineimmune response and to what extent through regulation of Tfh cell pathway. To address thesequestions, our study took advantage of IFN-a as an adjuvant with FMD recombinantadenovirus vaccine by investigating T follicular helper cells development, germinal center(GC) formation, B cell development, Tfh cells transcription factor Bcl-6mRNA expressionand Tfh cell related cytokine IL-21secretion to explore the role of IFN-a as an adjuvant howto regulate the immune responses with FMD adenovirus vaccine, providing a theoretical basisfor further study of IFN-a adjuvant as a new boost of vaccination responses.Methods: Female BALB/c mice used were4–6weeks of age.Each group was immunizedwith either108TCID50rAd5VP1or108TCID50rAd5VP1-2A-PoIFN-a, or108TCID50rAd5VP1+rAd5poIFN-a,and108TCID50Ad was negative control. Mice were injected oncei.p.rAd5VP1was made up with recombinant adenovirus expressing foot and mouth diseasevirus (FMDV) VP1protein, rAd5poIFN-a was made up with recombinant adenovirusexpressing porcine IFN-a and rAd5VP1-2A-PoIFN-a was made up with recombinantadenovirus co-expressing FMDV VP1-2A genes and porcine IFN-a.1The spleens from the BALB/c mice were harvested on day7after immunization, andsplenocytes were analyzed by flow cytometry to determine the frequency of CXCR5+PD-1+Tfh cells present within the total CD4+population. 2The spleens from the BALB/c mice were harvested on day7after immunization, andthe splenocytes were analyzed by flow cytometry to determine the frequency of theGL-7+B220+GC B cells.3The Splenocytes from BALB/c mice were harvested on day7after immunization, andtotal cellular RNA was prepared with an AxyPrep Total RNA Miniprep Kit (AxyGEN), andBcl-6mRNA expression was assessed by real-time reverse transcription polymerase chainreaction (RT-PCR) analysis.4The spleens from BALB/c mice on day7after immunization were fixed in4%paraformaldehyde and embedded in paraffin. Sections were cut to4μm thickness on a routineparaffin and stained for immunofluorescenceusing PNA-FITC for GCs.Mean Fluorescence Intensity analyzed by the Image J software.5Mice serum was harvested after vaccination on day7, cytokine IL-21was assayed bysandwich ELISA.Results:1IFN-a as an adjuvant to recombinant adenovirus vaccine can enhance the generation ofT follicular helper cells.The splenocytes were harvested on day7after immunization, and thedevelopment of CXCR5+PD-1+CD4+Tfh cells were determined by multiple-color flowCytometry. The percentages of CD4+CXCR5+Tfh cells within total CD4+T cells wereexamined and we found a obvious increase in the frequency of CXCR5+CD4+T cells in miceimmunized with adenoviral vectors co-expressing FMDV VP1proteins andIFN-a(2.66%±0.16) or combined immunodeficiency individual expression of FMDV VP1adenovirus (rAd5VP1) and porcine IFN-a recombinant adenovirus (rAd5poIFN-a) group(2.54%±0.44) compared with adenoviral vectors expressing VP1alone (1.29%±0.10)，P<0.05;the percentages of CD4+PD-1+Tfh cells within total CD4+T cells were examined and wefound a obvious increase in the frequency of PD-1+CD4+T cells in mice immunized withadenoviral vectors co-expressing FMDV VP1proteins and IFN-a (2.18%±0.38) or combined immunodeficiency individual expression of FMDV VP1adenovirus (rAd5VP1) and porcineIFN-a recombinant adenovirus (rAd5poIFN-a) group (2.22%±0.35) compared with adenoviralvectors expressing VP1alone (1.49%±0.13)， P<0.05; the percentages ofCD4+CXCR5+PD-1+Tfh cells within total CD4+T cells were examined and we found aobvious increase in the frequency of CXCR5+PD-1+CD4+T cells in mice immunized withadenoviral vectors co-expressing FMDV VP1proteins and IFN-a (2.22%±0.35) or combinedimmunodeficiency individual expression of FMDV VP1adenovirus (rAd5VP1) and porcineIFN-a recombinant adenovirus (rAd5poIFN-a) group (2.19%±0.37)compared with adenoviralvectors expressing VP1alone(1.49%±0.13)，P<0.05;2IFN-a as an adjuvant to recombinant adenovirus vaccine can enhance the generation ofgerminal center B cells.The splenocytes were harvested on day7after immunization, and thedevelopment of GL-7+B220+B cells were determined by multiple-color flow Cytometry. Thefraction of GL-7+B220+B cells were also observed and we found a significant increase of thispopulation in mice immunized with adenoviral vector co-expressing FMDV VP1proteins andIFN-a (7.39%±0.38) or combined immunodeficiency individual expression of FMDV VP1adenovirus (rAd5VP1) and porcine IFN-a recombinant adenovirus (rAd5poIFN-a) group(4.44%±0.35)compared with mice immunized with rAd5VP1alone (2.65%±0.28), P<0.05.3IFN-a as an adjuvant to recombinant adenovirus vaccine can improve the expression ofthe Tfh cell transcription factor Bcl-6.The spleens were harvested on day7,and Bcl-6mRNAexpression were analyzed by real-time RT-PCR. Consistent with above results, adenoviralvectors co-expressing FMDV VP1proteins and IFN-a group showed the significant upregulation of Bcl-6expression (10.47±1.54）,or combined immunodeficiency individualexpression of FMDV VP1adenovirus (rAd5VP1) and porcine IFN-a recombinant adenovirus(rAd5poIFN-a) group (9.65±1.79)compared with adenoviral vectors expressing FMDV VP1proteins alone (3.42±0.27), P <0.01.4IFN-a as an adjuvant to recombinant adenovirus vaccine can increase the formation of germinal center.The spleens were harvested on day7,and GCs were measured by histologicalstaining of lymphoid tissue using the lectin PNA. The generation of GCs were greatlyincreased in mice immunized with co-expression of FMDV VP1proteins and IFN-a(2.41±0.32) compared with mice immunized with rAd5VP1alone （1.39±0.07）, P<0.01,aswell as combined immunodeficiency individual expression of FMDV VP1adenovirus(rAd5VP1) and porcine IFN-a recombinant adenovirus (rAd5poIFN-a) group (2.13±0.13)，P <0.05,which was consistent with our flow Cytometric analysis.5IFN-a as an adjuvant to recombinant adenovirus vaccine can increase the production ofIL-21. One day7after the immunization, the sera were collected and assayed by ELISA forthe measurement of IL-21.ELISA results showed that rAd5VP1-2A-PoIFN-a(149.50±32.74,pg/ml) could enhance IL-21production, compared with rAd5Vp1（63.68±13.15，pg/ml）,P<0.01. Meanwhile,rAd5VP1+rAd5poIFN-a group (130.60±19.80，pg/ml) also showed the increase of IL-21production compared with rAd5Vp1, P<0.05.Conclusions: Based on our results, the conclusions could be drawn that IFN-a as anadjuvant to recombinant FMD adenovirus vaccine can enhance T follicular helper cellsdevelopment, germinal center formation, B cell development, Tfh cells transcription factorBCL-6mRNA expression and Tfh cell related cytokine IL-21secretion after Immunization onday7. Meanwhile our data also showed that the IFN-a as an adjuvant can improvedifferentiation and proliferation of Tfh cells during the immune responses with FMDadenovirus vaccine, these results could contribute to the theoretical basis of IFN-a adjuvant asa novel boost of vaccination responses. |
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