Critical Role of Perforin-dependent CD8+ T Cell Immunity for Rapid Protective Vaccination in a Murine Model for Human Smallpox

Kremer, Melanie; Suezer, Yasemin; Volz, Asisa; Frenz, Theresa; Majzoub, Monir; Hanschmann, Kay-Martin; Lehmann, Michael H.; Kalinke, Ulrich; Sutter, Gerd

Thus, we depleted C57BL/6 mice of CD4+ T cells, CD8+ T cells or both T cell subsets by i.p. injection of specific antibodies, and confirmed successful depletion by FACS analysis at the time point of MVA immunization (day 0) and at day 7 after vaccination (Figure S6B,C; data not shown). Control C57BL/6 mice were again fully protected by MVA immunization two days prior to the lethal respiratory challenge infection with ECTV ( Figure 4A ). In contrast, vaccinated mice depleted of CD8+ T cells, or both T cell subsets ( Figure 4B ,C) succumbed to ECTV infection, with similar disease pattern and time to death as compared to unvaccinated animals. On the other hand, depletion of CD4+ T cells ( Figure 4D ) prior to MVA immunization resulted in delayed onset of disease, since the start of striking body weight loss occurred about six days after the onset of symptoms in unvaccinated controls. Nevertheless, CD4+ T cell depletion in immunized animals also resulted in 100% mortality within 21 days post challenge. These data clearly suggested that both CD4+ and CD8+ T cells are required to rapidly protect mice by MVA vaccination. To assess the need for CD4+ T cells in some more detail, we analyzed CD4-depleted C57BL/6 mice for defects in mounting VACVspecific antibodies or CD8+ T cells following MVA immunization. Lack of CD4+ T cells resulted only in a minor reduction of IgG antibody responses as revealed by ELISA testing of sera at day 21 post vaccination with 10 8 PFU MVA (i.n.). The CD4 cell depleted mice were clearly able to mount levels of VACVspecific antibodies (mean titer of pooled sera 1280) that were just about two fold lower than responses obtained in control mice (data not shown). To study the impact of CD4+ T cell depletion on MVA induced CD8+ T cell responses we monitored for the expansion of endogenous CD8+ T cells specifically recognizing the B8R 20-27 epitope (TSYKFESV) by FACS analysis using a TSYKFESV-Kb pentamer (ProImmune) ( Figure 5 ). Upon inoculation of C57BL/6 mice with 2610 5 PFU MVA, B8R-specific T cells massively expanded and reached a maximum of approximately 20% of total CD8+ T cells at day 6 after infection. On the contrary, MVA-vaccinated CD4+ depleted mice showed a significantly reduced CD8+ T cell expansion reaching less than 10% of total CD8+ T cells ( Figure 5A ). This data suggested that CD4+ T cells seem to play an important role in regulating the strength of the MVA induced CD8-T cell response.