During the effector phase of a virus-specific adaptive immune response, CD8+ T cells migrate to the target organ of infection. The cytotoxic T cell (CTL) response to pathogens is directed against antigenic epitopes, that are processed by proteasomes and presented by MHC class I molecules. Immunoproteasomes have been shown to optimize the quantity and quality of peptide ligands in several infection models. Due to the finding that a potent CD8+ T cell response is important to mediate CVB3 virus elimination (33;126), it was of great interest to investigate the effect of β1i/LMP2- and β5i/LMP7-deficiency on CD8+ T cell immunity in CVB3 myocarditis. Previous in vitro processing studies demonstrated, that cardiac IP formation is linked to the preferential generation of CVB3-specific epitopes as compared to standard proteasomes (118).
A B
P3D [2170-2177] VP2 [285-293]
0.0 0.2 0.4 0.6 0.8
1.0 control
CVB3 d8 p.i.
*
*
% of pentamer-positive CD8+ T cells
Fig. 10 Frequencies of CVB3 epitope-specific T cells in acute myocarditis. CD8+ T cells isolated from naive and CVB3-infected C57BL/6 mice (d8 p.i.) were stained with H-2Db VP2 [285-293]-PE and H-2Kb P3D
[2170-To test the potential of these epitopes to elicit an antigen specific CD8+ T cell response in vivo, CD8+ T cell were isolated from sham-treated and CVB3-infected C57BL/6 mice and stained with epitope-specific P3D [2170-2177] and VP2 [285-293] phycoerythrin (PE)-coupled pentamers.
FACS analysis revealed a CVB3-specific response in infected C57BL/6 mice, that was predominantly directed against P3D [2170-2177] and to a lesser extend against VP2 [285-293] (Fig. 10B).
5.2.1 CD8
+T cell function
Overall P3D- and VP2-specific CD8+ T cell frequencies were rather low (<1 %, see Fig. 10) and the existence of other, so far unknown epitopes could not be excluded. To study whether IP-deficiency has an impact on the function of CD8+ T cells despite these limitations, adoptive transfer of cytotoxic CD8+ T cells was performed. For this purpose, CD8+ T cells were isolated from total splenocytes of CVB3-infected C57BL/6 wildtype, β1i/LMP2-/- and β5i/LMP7-/- donor mice, respectively. MACS separation resulted in at least 89 % purity of CD8+ T cells as representatively shown in Fig. 11.
Fig. 11 Purity of CD8+ T cells. CD8+ T cells were isolated from total splenocytes of CVB3-infected animals by MACS separation. After fluorescent labeling of cells with anti-CD3-FITC and anti-CD8-PB antibodies, purity was determined by flow cytometry. Representative dot blots indicate the percentage of CD3+/CD8+ T cells prior and after MACS separation.
To preclude effects of IP-deficiency on T cell survival, CD8+ T cells from CVB3-challenged β5i/LMP7-/- and β1i/LMP2-/- mice (both CD45.2) were transferred into B6.SJL-Ptprca Pepcb/BoyJ mice (CD45.1) and vice versa. After transfer, recipient mice were immediately infected, and the amount of donor CD8+ T cells was determined at d8 p.i. Therefore, total splenocytes were stained with CD8-PB and CD45.1-PerCPCy5 respectively anti-CD45.2-FITC antibodies and analyzed by flow cytometry.
A
% of donor CD8+ T cells in recipient mice
C57BL/6
% of donor CD8+ cells in recipient mice
D E mice were infected with CVB3 and the survival of donor CD45.1 and CD45.2 T cells was determined by FACS
CD8 positive T cells were gated as illustrated in Fig. 12A and the fraction of donor CD8+/CD45+ T cells was determined.
Deficiency of β5i/LMP7 had no effect on T cell survival in the respective recipients (Fig.
12B). By contrast, donor CD8+ T cells from β1i/LMP2-deficient mice showed decreased survival rates as compared to IP-competent lymphocytes (Fig. 12C).
To get more functional insights concerning CD8+ T cell responses with respect to the target organ of CVB3 infection, heart tissue injury was assessed after adoptive transfer of CD8+ T cells by hematoxylin-eosine staining. Myocarditis scores of β5i/LMP7-competent and β5i/LMP7-deficient recipient mice were found to be within the same range, independent from the origin of donor CD8+ T cells (Fig. 12D). By contrast, myocarditis scores of β1i/LMP2 -/-recipient mice were significantly lower compared to C57BL/6 -/-recipients (Fig. 12E).
However, no protective or deleterious effects on the extent of acute myocardial injury were observed in these recipients upon transfer of β1i/LMP2-competent or β1i/LMP2-deficient CD8+ T cells.
5.2.2 Determination of CVB3-specific IgG titers by ELISA
B cells isolated from C57BL/6 mice preferentially express IPs (127), and a CVB3-specific IgG antibody response contributes to an efficient virus elimination in CVB3 myocarditis (128). Therefore, it was important to characterize the function of IPs for the induction of a systemic B cell response in CVB3 myocarditis. CVB3-specific antibodies IgG titers were determined in sera of C57BL/6, β5i/LMP7-/- andβ1i/LMP2-/- mice at d8 and d28 p.i.
Fig. 13 Determination of CVB3-specific antibody titers. Titers of virus-specific IgG antibodies were assessed in sera of C57BL/6, β5i/LMP7-/- and β1i/LMP2-/- mice at indicated time points by a CVB3-specific ELISA (n≥8+SEM).
Serum levels of anti-CVB3 IgG antibodies were within the same range in all three investigated strains at d8 p.i. (Fig. 13). By day 28 p.i., an increase of IgG titers was observed,
whereby no strain-specific differences were detected, which demonstrated that β5i/LMP7-and β1i/LMP2-deficient mice were capable of generating an efficient high affinity antibody response.
5.2.3 Characterization of splenic cell subpopulations by FACS analysis
As mentioned before, immune cells, including T and B cells as well as professional APCs such as dendritic cells, constitutively express IPs (129;130). To investigate whether IP expression is important for the maintenance of homeostatic T and B cell levels, the percentage of splenic CD3+/CD4+, CD3+/CD8+ T cells, and CD19+/B220+ B cells was determined by FACS analysis over the course of CVB3 infection. The relative amount of CD4+ T, CD8+ T and B cells was not affected in sham-treated or CVB3-infected β5i/LMP7-/- mice as compared to C57BL/6 mice.Fig. 14 FACS analysis of total splenocytes. Single splenocyte suspensions of C57BL/6, β5i/LMP7-/- and β1i/LMP2-/- mice were prepared and the percentage of splenic subpopulations was determined by flow cytometry at d0, d4 and d8 p.i. A: CD3+/CD4+, CD3+/CD8+ T cells, and CD19+/B220+ B cells were gated as demonstrated and B: the the relative amount of these subpopulations was determined (n≥8 mice/group,*p<0.05).
the relative number of CD4+ T cells as well as B cells were observed as compared to C57BL/6 wildtype mice (Fig. 14B).
Interestingly, quantification of total splenocytes resulted in significantly lower absolute numbers in naive β1i/LMP2-/- control mice as compared with C57BL/6 and β5i/LMP7-/- control mice (Fig. 15A). Upon CVB3 infection, a 3-fold increase in total splenocytes was observed in β1i/LMP2-/- mice in contrast to C57BL/6 and β5i/LMP7-/- mice at d8 p.i.
Absolute numbers of splenic subpopulations were determined in naive mice by multiplying splenic frequencies by the total cell number (per spleen). As illustrated in Fig. 15B, naive β1i/LMP2-/- mice had significantly reduced baseline numbers of splenic CD4+, CD8+ and B cells. Splenic hyperplasia as observed upon CVB3 infection in β1i/LMP2-/- mice explains equal relative amounts of CD8+ T cells in β1i/LMP2-/- mice compared to C57BL/6 mice at d8 p.i. (Fig. 7 middle panel).
C57BL/6β1i/LMP2 -/- CD4 CD8
0
-/-Fig. 15 Quantification of absolute splenocyte numbers in naive mice. A: The absolute number of splenocytes was determined by counting total splenocytes. B: Subsequent calculation of CD3+/CD4+, CD3+/CD8+ T cell and CD19+/B220+ B cell numbers based on ratios from FACS analysis by multiplying splenic frequencies by the total number of cells from each spleen (n≥4, *p<0.05).