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. 2014 Apr;25(4):350-63.
doi: 10.1089/hum.2013.218. Epub 2014 Mar 31.

Adenovirus vector-induced CD8⁺ T effector memory cell differentiation and recirculation, but not proliferation, are important for protective immunity against experimental Trypanosoma cruzi Infection

José Ronnie Vasconcelos  1 , Mariana R Dominguez, Ramon L Neves, Jonatan Ersching, Adriano Araújo, Luara I Santos, Fernando S Virgilio, Alexandre V Machado, Oscar Bruna-Romero, Ricardo T Gazzinelli, Mauricio M Rodrigues
Affiliations

Adenovirus vector-induced CD8⁺ T effector memory cell differentiation and recirculation, but not proliferation, are important for protective immunity against experimental Trypanosoma cruzi Infection

José Ronnie Vasconcelos et al. Hum Gene Ther. 2014 Apr.

Abstract

Heterologous prime-boost vaccination using plasmid DNA followed by replication-defective adenovirus vector generates a large number of specific CD8⁺ T effector memory (TEM) cells that provide long-term immunity against a variety of pathogens. In the present study, we initially characterized the frequency, phenotype, and function of these T cells in vaccinated mice that were subjected to infectious challenge with the human protozoan parasite Trypanosoma cruzi. We observed that the frequency of the specific CD8⁺ T cells in the spleens of the vaccinated mice increased after challenge. Specific TEM cells differentiated into cells with a KLRG1(High) CD27(Low) CD43(Low) CD183(Low)T-bet(High) Eomes(Low) phenotype and capable to produce simultaneously the antiparasitic mediators IFNγ and TNF. Using the gzmBCreERT2/ROSA26EYFP transgenic mouse line, in which the cells that express Granzyme B after immunization, are indelibly labeled with enhanced yellow fluorescent protein, we confirmed that CD8⁺ T cells present after challenge were indeed TEM cells that had been induced by vaccination. Subsequently, we observed that the in vivo increase in the frequency of the specific CD8⁺ T cells was not because of an anamnestic immune response. Most importantly, after challenge, the increase in the frequency of specific cells and the protective immunity they mediate were insensitive to treatment with the cytostatic toxic agent hydroxyurea. We have previously described that the administration of the drug FTY720, which reduces lymphocyte recirculation, severely impairs protective immunity, and our evidence supports the model that when large amounts of antigen-experienced CD8⁺ TEM cells are present after heterologous prime-boost vaccination, differentiation, and recirculation, rather than proliferation, are key for the resultant protective immunity.

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Figures

<b>FIG. 1.</b>
FIG. 1.
Frequencies of specific splenic CD8+ T cells increase after infectious challenge with T. cruzi in mice vaccinated with the heterologous prime-boost vaccination regimen. (A) C57BL/6 mice were immunized and challenged as depicted. Priming and boosting immunizations were performed as described in the Materials and Methods section. Mouse groups: naïve, nonmanipulated; β-Gal/T. cruzi, immunized with the control vector pcDNA3/Adb-Gal and challenged with T. cruzi; ASP-2, immunized with pIgSP Cl.9/AdASP-2; ASP-2/T. cruzi, immunized pIgSP Cl.9/AdASP-2 and challenged with T. cruzi. (B) H-2Kb-VNHRFTLV+CD8+ splenic cell frequencies were estimated in pools of splenic cells from 4 mice. (C) GzmBCreERT2/ROSA26EYFP transgenic mice were immunized and challenged as depicted. (D) H-2Kb-VNHRFTLV+CD8+ cell frequencies were estimated in the spleen of individual mouse (n=4). (E) The total number of H-2Kb-VNHRFTLV+CD8+ cells was estimated in the spleen of each individual mouse (n=4). (F) H-2Kb-VNHRFTLV+CD8+ cell frequencies were estimated in the inguinal and popliteal lymph nodes of each individual mouse. (G) The total numbers of H-2Kb-VNHRFTLV+CD8+ cells were estimated in the inguinal and popliteal lymph nodes of each individual mouse (n=4). The dots represent each mouse, and the bars represent the means±SD.
<b>FIG. 2.</b>
FIG. 2.
Frequencies of specific cytokine-secreting splenic CD8+ T cells increase after infectious challenge with T. cruzi in mice vaccinated with the heterologous prime-boost vaccination regimen. (A) C57BL/6 mice were immunized and challenged as depicted. Priming and boosting immunizations were performed as described in the Materials and Methods section. The mouse groups were the same as those described in the legend of Fig. 1. (B) Pools of splenic cells from 4 mice were restimulated in vitro in the absence (Med) or presence of the peptide VNHRFTLV (Pep), anti-CD28, BdGolgiPlug, and monensin. After 12 hr, the cells were stained with anti-CD8, anti-IFNγ, and anti-TNF. (C) The same as above expect that they represent only the cells stained with anti-CD8 and anti-IFNγ but not with anti-TNF. (D) C57BL/6 mice were immunized and challenged as depicted. (E) Splenic cells were restimulated in vitro in the presence of the peptide VNHRFTLV, anti-CD28, BdGolgiPlug, and monensin. After 12 hr, the cells were stained with anti-CD8, anti-IFNγ, and anti-TNF. The results are represented by medians (bars) from four individual mouse per group (dots). Two asterisks denote that the values of these groups were higher than those of the naïve control mice or all other groups (p<0.01), respectively. (F) Pie charts show the fraction of peptide-specific cells expressing the indicated molecules. The results are expressed as the mean values for four mice per group.
<b>FIG. 3.</b>
FIG. 3.
Phenotype of specific splenic CD8+ T cells after infectious challenge with T. cruzi in mice vaccinated with the heterologous prime-boost vaccination regimen. (A) C57BL/6 mice were immunized and challenged as depicted. Priming and boosting immunizations were performed as described in the Materials and Methods section. The mouse groups were the same as those described in the legend of Fig. 1. The red and blue lines depict the naïve CD8+ cells and H-2Kb-VNHRFTLV CD8+ cells, respectively. Pools of splenic cells from 4 mice were stained with anti-CD8, H-2Kb-VNHRFTLV, and anti-KLRG, anti-CD27, anti-CD43, or anti-CD183. The red and blue lines denote the naïve CD8+ cells and H-2Kb-VNHRFLTV CD8+ cells, respectively. Analysis of individual mouse in each group provided similar results. (B) Pools of splenic cells from four mice were stained with anti-CD8, H-2Kb-VNHRFTLV, and anti-T-bet or anti-Eomes. (C) Boolean analyses of pools of splenic cells from 4 mice that were simultaneously stained with anti-CD8, H-2Kb-VNHRFLTV, anti-KLRG, anti-CD27, anti-CD43, and anti-CD183. The red and blue lines denote the naïve CD8+ cells and H-2Kb-VNHRFLTV CD8+ cells, respectively. Analysis of individual mouse in each group provided similar results.
<b>FIG. 4.</b>
FIG. 4.
The specific CD8+ T cells detected after challenge with T. cruzi are TE(M) cells. (A) GzmBCreERT2/ROSA26EYFP transgenic mice were immunized, treated with tamoxifen, and challenged as depicted. The mouse groups were the same as those described in the legend of Fig. 1. (B) Splenocytes were stained with anti-CD8 and H-2Kb-VNHRFTLV. The EYFP+ TE cell frequency in each group was then estimated. (C) Splenocytes isolated at day 52 were stained with anti-CD8 and H-2Kb-VNHRFTLV. The frequency of EYFP+ cells (Granzyme B+ TE) in each group is shown for each individual mouse (dots). Bars represent the median of each mouse group. (D) The same as above except that the cells were obtained from mice at day 102. The asterisk denotes a higher frequency of EYFP+ H-2Kb-VNHRFTLV + CD8+ cells (p<0.05).
<b>FIG. 5.</b>
FIG. 5.
EYFP+ H-2Kb-VNHRFTLV+ and EYFP+ H-2Kb-VNHRFTLV− CD8+ T cell frequencies increase after infectious challenge. (A) GzmBCreERT2/ROSA26EYFP transgenic mice were immunized, treated with tamoxifen, and challenged as depicted. The mouse groups were the same those as described in the legend of Fig. 1. M1–M4 denote each individual mouse analyzed. (B) The frequency of the splenic H-2Kb-VNHRFTLV+ CD8+ cells was higher in the ASP-2/T. cruzi mice compared with the ASP-2 mice (p<0.01). (C) Frequencies of the EYFP+ H-2Kb-VNHRFTLV+ and EYFP+ H-2Kb-VNHRFTLV− CD8+ cells in the ASP-2 and ASP-2/T. cruzi mice. (D) Frequencies of EYFP+ H-2Kb-VNHRFTLV+ CD8+ T cells/total EYFP+ CD8+ T cells. No significant difference was found between the frequency of the EYFP+ H-2Kb-VNHRFTLV+ CD8+ cells in the ASP-2/T. cruzi mice compared with that of the ASP-2 animals (p=NS). Results are from one of three independent experiments performed.
<b>FIG. 6.</b>
FIG. 6.
The increase in the specific splenic CD8+ T cell frequency after infectious challenge is not inhibited by treatment with hydroxyurea (HU). (A) C57BL/6 mice were immunized, infected, and treated with HU as depicted. The mouse groups were the same as those described in the legend of Fig. 1 except that βGal denotes mice that were injected with pcDNA3 and Adβ-Gal. (B) Fourteen days after challenge, the H-2Kb-VNHRFTLV+ CD8+ cell frequency was estimated. Representative plot of each group. (C) Number of splenic H-2Kb-VNHRFTLV+ CD8+ cell expressed as mean±SD (n=4). Results are from one of two independent experiments performed.
<b>FIG. 7.</b>
FIG. 7.
The protective immunity elicited by heterologous prime-boost vaccination is not inhibited by treatment with HU. (A) A/Sn mice were immunized, infected, and treated with HU as depicted. (B) Parasitemia of each individual control mouse primed with pcDNA3 and boosted with Adβ-Gal (n=12). (C) Parasitemia of each individual ASP-2-immunized mouse (n=12). (D) Parasitemia of each individual ASP-2-immunized mouse treated with HU (n=10). Although the day of peak parasitemia was different for each mouse, we used the maximal values for statistical comparison. The results of the statistical comparisons are below panel D. (E) Kaplan–Meier survival curves of the different groups were compared, and the results showed that ASP-2-immunized mice treated or not with HU survived significantly longer (p<0.01) than control animals injected with βGal. Results are from two experiments pooled.
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