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. 2023 Dec;22(12):e14007.
doi: 10.1111/acel.14007. Epub 2023 Nov 23.

Depletion of preexisting B-cell lymphoma 2-expressing senescent cells before vaccination impacts antigen-specific antitumor immune responses in old mice

Ozmen Cobanoglu  1 , Lou Delval  1 , Daniele Ferrari  2 , Lucie Deruyter  1 , Séverine Heumel  1 , Isabelle Wolowczuk  1 , Abir Hussein  3 , Ayse Nur Menevse  3 , David Bernard  4 , Philip Beckhove  3   5 , Frauke Alves  2 , François Trottein  1
Affiliations

Depletion of preexisting B-cell lymphoma 2-expressing senescent cells before vaccination impacts antigen-specific antitumor immune responses in old mice

Ozmen Cobanoglu et al. Aging Cell. 2023 Dec.

Abstract

The age-related decline in immunity reduces the effectiveness of vaccines in older adults. Immunosenescence is associated with chronic, low-grade inflammation, and the accumulation of senescent cells. The latter express Bcl-2 family members (providing resistance to cell death) and exhibit a pro-inflammatory, senescence-associated secretory phenotype (SASP). Preexisting senescent cells cause many aging-related disorders and therapeutic means of eliminating these cells have recently gained attention. The potential consequences of senescent cell removal on vaccine efficacy in older individuals are still ignored. We used the Bcl-2 family inhibitor ABT-263 to investigate the effects of pre-vaccination senolysis on immune responses in old mice. Two different ovalbumin (OVA)-containing vaccines (containing a saponin-based or a CpG oligodeoxynucleotide adjuvant) were tested. ABT-263 depleted senescent cells (apoptosis) and ablated the basal and lipopolysaccharide-induced production of SASP-related factors in old mice. Depletion of senescent cells prior to vaccination (prime/boost) had little effect on OVA-specific antibody and T-cell responses (slightly reduced and augmented, respectively). We then used a preclinical melanoma model to test the antitumor potential of senolysis before vaccination (prime with the vaccine and OVA boost by tumor cells). Surprisingly, ABT-263 treatment abrogated the vaccine's ability to protect against B16 melanoma growth in old animals, an effect associated with reduced antigen-specific T-cell responses. Some, but not all, of the effects were age-specific, which suggests that preexisting senescent cells were partly involved. Hence, depletion of senescent cells modifies immune responses to vaccines in some settings and caution should be taken when incorporating senolytics into vaccine-based cancer therapies.

Keywords: Bcl-2; aging; cellular senescence; immune responses; senolytics; tumor growth; vaccination.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Depletion of senescent cells by ABT‐263 and consequences on LPS‐induced production of SASP‐related cytokines by old splenocytes. (a) Splenocytes collected from young and old mice were incubated with the fluorescent β galactosidase substrate CF12FDG and detection of the fluorescent substrate cleaved by β‐galactosidase was performed by flow cytometry. Representative profiles are depicted (upper panel: the CD45‐positive fraction and lower panel: the CD45‐negative fraction). (b) Left panel, Old splenocytes were cultured in the presence of ABT‐263 (1 μM) for 24 h and SA‐β‐galactosidase activity was assessed by flow cytometry. The percentages of labelled cells are indicated. (c) Frequencies of annexin V positive cells among each cell population (4 h post ABT‐263 treatment). (d) Splenocytes were treated with vehicle or ABT‐263 (1 μM) for 24 h and then stimulated with LPS at 1 μg/mL. After 24 h, supernatants were collected and cytokine production was determined by ELISA. For all graphs, errors indicate mean (n = 3–4). One representative experiment out of two (a–c) or three (d) are depicted. Significant differences were determined using the unpaired t test (b and c) and the two‐way ANOVA Tukey's multiple comparisons test (d) (*p < 0.05; **p < 0.01; ***p < 0.01).
FIGURE 2
FIGURE 2
Effect of ABT‐263 treatment in the SASP signature in old mice. (a) Schematic procedure. Young and old mice were orally treated with ABT‐263 for 4 days. One day (Day 5) and 4 days later (Day 8), cell apoptosis and cellular senescence were respectively analyzed. (b) Representative images of SA‐β‐Gal staining of spleen sections. Arrows depict positive cells. (c) Representative images of cleaved caspase‐3 (Day 5) and p16, Bcl‐2, and Bcl‐xl (Day 8) staining of spleen sections. Arrows depict positive cells. (d) The percentages of PD1+ and CD153+ cells within CD4+ CD44high CD62Llow cells are depicted for the four animal groups (n = 6–12) (Day 8). (e) Frequencies of annexin V positive cells among CD45+ cells (n = 3) (Day 5). (f) Cytokines in blood were quantified by ELISA (n = 3–5) (Day 8). (g) Splenocytes (Day 8) were stimulated with LPS (1 μg/mL). Supernatants were collected and cytokine production was determined by ELISA (n = 7). Pooled results from two independent experiments (d and g) and one of two representative experiments (b, c, e and f) are shown. Significant differences were determined using two‐way ANOVA Tukey's multiple comparisons test (*p < 0.05; **p < 0.01; ***p < 0.01).
FIGURE 3
FIGURE 3
Effects of ABT‐263 treatment on humoral and cellular OVA‐specific immune responses in old and young mice. (a) Upper panel, schematic procedure (prime‐boost setting). Mice were orally treated with ABT‐263 for 4 days. After a 4‐day interval, the procedure was repeated. Three days after the last ABT‐263 inoculation, mice were immunized with the vaccine (prime). Animals were boosted 7 days later and sacrificed 13 days after the boost. Lower panel, Body weight was measured over the course of treatment and vaccine procedure (n = 5). (b) IL‐6 concentration in blood was quantified by ELISA (n = 5). (c and d) IgM and IgG titers were determined by indirect ELISA. Serum samples were collected after the prime (c) and after the boost (d) (n = 5). (e) LN cells and spleen cells from vaccinated mice were restimulated with whole OVA or OVA257–264, for 48 h. IFN‐γ production was assessed by ELISA (n = 5). (a–e) One representative experiment out of two performed are depicted. Significant differences were determined using the two‐way ANOVA Tukey's multiple comparisons test (*p < 0.05).
FIGURE 4
FIGURE 4
Effects of ABT‐263 treatment on OVA‐specific immune responses and B16‐OVA growth in young and old mice. (a) Schematic procedure (prime‐only regimen and challenge with B16‐OVA). Mice were orally treated with ABT‐263 for 4 days. After a 4‐day interval, the procedure was repeated. Three days after the last ABT‐263 inoculation, mice were immunized with the vaccine. Tumor cells were inoculated 10 days later and sacrificed 23 days after the inoculation. (b) IgM, IgG, IgG1, and IgG2a titers were determined by indirect ELISA (dilution: 1:800) (n = 3 for unvaccinated mice and n = 5 for vaccinated mice). (c) LN cells and spleen cells from vaccinated young and old mice were restimulated with OVA or OVA257–264 for 48 h. Supernatants were collected and IFN‐γ were quantified by ELISA (n = 10). (d) B16‐OVA tumor volumes were measured over time (n = 6 for unvaccinated mice and n = 10 for vaccinated mice). (a and b) A representative experiment out of two is depicted. (c and d) A pool of two representative experiments is depicted. Significant differences were determined using the two‐way ANOVA Tukey's multiple comparisons test (*p < 0.05; **p < 0.01; ***p < 0.01).
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