Enhancement of humoral and cellular immunity with an anti-glucocorticoid-induced tumour necrosis factor receptor monoclonal antibody

Abstract

Adjuvants, including antibodies to tumour necrosis factor receptor superfamily members, augment immune responses. One member of this family, glucocorticoid-induced tumour necrosis factor receptor (GITR), is expressed at low levels on naive/resting T cells, B cells and macrophages, but at higher levels on T regulatory cells. The aim of this study was to determine the ability of a rat anti-mouse GITR monoclonal antibody, 2F8, to stimulate murine humoral and cellular immunity in a prime boost model with particular attention to posology and antigen-specific effects. 2F8 enhanced the humoral immune response to ovalbumin and haemagglutinin (HA) compared with controls and this enhancement was equal to or greater than that obtained in mice dosed with standard adjuvants. 2F8 F(ab')(2) fragments were as effective as intact antibody in boosting humoral immunity, indicating that FcR-mediated cross-linking of 2F8 is not required for efficacy. Moreover, the enhanced response was durable and antigen specific. Administration of 2F8 shifted the immune response towards a T helper type 1 response with significant enhancement of immunoglobulin G2a- and G2b-specific anti-HA antibodies, as well as enhanced cellular immunity as measured by ELISPOT. 2F8-treated mice also generated significantly more neutralizing antibodies to HA than control mice. Our findings show that anti-GITR is a robust, versatile adjuvant that, unlike commonly used adjuvants that primarily enhance humoral immunity, enhances both humoral and cellular immunity. These results support the continued development of anti-GITR for such indications as haematological and solid tumours, chronic viral infections, and as a vaccine adjuvant.

Figure 1

2F8 enhances humoral immunity to ovalbumin (OVA). Antibody dosing and immunization schedule are indicated above the graph. OVA mixed with alum or incomplete Freund’s adjuvant (IFA) is included for comparison. Levels of anti-OVA antibodies were determined by enzyme-linked immunosorbent assay as described in the Materials and methods section. Titre is defined in the Materials and methods section. Results represent at least two independent experiments with three to five mice per group. All treated groups were significantly higher than antibody isotype control-treated mice. Significant comparisons are shown, all others were not significant. ***P < 0·001, **P < 0·01, *P < 0·05.

Figure 2

2F8-enhanced humoral immunity to ovalbumin (OVA) is durable and does not require Fc–FcR interactions. Antibody dosing and immunization schedules are indicated above the graph; 8 mg of 2F8 F(ab′)₂ fragments (a and b), 0·4 mg 2F8 (a, b, and c), or 0·4 mg YT765 (c) were used in the studies. Anti-OVA titres determined by enzyme-linked immunosorbent assay were analysed on day 21 (a and c) and day 63 (b). Results represent at least two independent experiments with three to five mice per group. Titres are compared against both the antigen-only and isotype control groups. **P < 0·001, *P < 0·01.

Figure 3

2F8 enhances humoral immunity to (a) H3N2 haemagglutinin (HA) and (b) H5N1 HA. Antibody dosing and immunization schedules are indicated above the graph; 0·4 mg 2F8 was used in these studies. HA mixed with alum or incomplete Freund’s adjuvant (IFA) is included for comparison. Levels of anti-HA antibodies were determined by enzyme-linked immunosorbent assay, as described in the Materials and methods section. Results represent at least two independent experiments with three to five mice per group. In (a), 2F8 is compared against both antigen-only and isotype control groups. **P < 0·01, *P < 0·05.

Figure 4

2F8-enhanced humoral immunity is specific for the antigen administered during antibody dosing. Antibody dosing and immunization schedules are indicated above the graph; 0·4 mg 2F8 was used in these studies. Mice were allowed to rest for 2 weeks before immunization with the neo-antigen, ovalbumin (OVA). No additional antibody was administered to any of the mice. Anti-haemagglutinin (HA) (a) and anti-OVA (b) titres were determined by enzyme-linked immunosorbent assay on day 56. Results represent at least two independent experiments with three to five mice per group. *P < 0·001.

Figure 5

The effect of the timing of antibody administration on humoral immunity. Antibody dosing and immunization schedule are indicated above the graph. Mice received 0·4 mg 2F8 either during the primary immunization or during the antigen challenge. Anti-haemagglutinin (HA) titres were determined by enzyme-linked immunosorbent assay on day 21. Titres of mice treated with 2F8 on days − 1, 0 and 1 versus mice treated on days 13, 14 and 15 are shown. Mice treated with a mixture of incomplete Freund’s adjuvant (IFA) and HA are included for comparison. Results represent at least two independent experiments with three to five mice per group. **P < 0·01, *P < 0·05.

Figure 6

2F8 enhances T helper type 1 humoral immunity; 0·4 mg 2F8 was used in these studies. Haemagglutinin (HA) mixed with incomplete Freund’s adjuvant (IFA) is included for comparison. Levels of immunoglobulin G1 (IgG1) -specific (a), IgG2a-specific (b) and IgG2b-specific (c) anti-HA antibodies were determined by enzyme-linked immunosorbent assay, as described in the Materials and methods section. Results represent at least two independent experiments with three to five mice per group. In (a), comparison is to the antigen-only group. In (b) and (c), 2F8 is compared with the antigen-only and IFA groups. ***P < 0·001, **P < 0·01, *P < 0·05.

Figure 7

2F8 augments cellular immunity. Total splenocytes from mice treated with saline, incomplete Freund’s adjuvant (IFA), or 2F8 were prepared on day 21, cultured with or without haemagglutinin (HA), and assessed for the number of interferon-γ-secreting cells. Results represent at least two independent experiments with three to five mice per group. *P < 0·001.

Figure 8

2F8 administration augments neutralizing antibodies to haemagglutinin (HA). Serum samples from day 21 bleeds were analysed for neutralizing antibodies to HA. Results represent at least two independent experiments with three to five mice per group. 2F8 is compared with antigen-only and isotype control groups. *P < 0·001.