Abatacept decreases disease activity in a absence of CD4(+) T cells in a collagen-induced arthritis model

Abstract

Introduction: Abatacept is a fusion protein of human cytotoxic T-lymphocyte-associated protein (CTLA)-4 and the Fc portion of human immunoglobulin G1 (IgG1). It is believed to be effective in the treatment of rheumatoid arthritis by inhibiting costimulation of T cells via blocking CD28-B7 interactions as CTLA-4 binds to both B7.1 (CD80) and B7.2 (CD86). However, the interaction of CD28 with B7 molecules is crucial for activation of naive cells, whereas it is unclear whether the action of already activated CD4(+) T cells, which are readily present in established disease, also depends on this interaction. The aim of this study was to determine whether the mode of action of abatacept depends solely on its ability to halt T cell activation in established disease.

Methods: Arthritis was induced in thymectomized male DBA/1 mice by immunisation with bovine collagen type II. The mice were subsequently depleted for CD4(+) T cells. Abatacept or control treatment was started when 80 % of the mice showed signs of arthritis. Arthritis severity was monitored by clinical scoring of the paws, and anti-collagen antibody levels over time were determined by enzyme-linked immunosorbent assay.

Results: Treatment with abatacept in the absence of CD4(+) T cells resulted in lower disease activity. This was associated with decreasing levels of collagen-specific IgG1 and IgG2a antibodies, whereas the antibody levels in control or CD4(+) T cell-depleted mice increased over time.

Conclusions: These results show that abatacept decreased disease activity in the absence of CD4(+) T cells, indicating that the mode of action of abatacept in established arthritis does not depend entirely on its effects on CD4(+) T cell activation.

Fig. 1

Abatacept decreased disease activity in mice depleted of CD4⁺ T cells by GK1.5. a Collagen-induced arthritis was induced in male DBA/1 mice. When 80 % of the mice showed signs of arthritis, treatment was started. One day before the start of treatment, CD4⁺ T cells were depleted by intraperitoneal administration of GK1.5, and the depletion was continued until the end of follow-up. Treatment was administered by intraperitoneal injection of either PBS (diamonds), GK1.5 (CD4 depletion) (squares), abatacept (circles) or a combination of GK1.5 and abatacept (triangles). The mice were scored three times per week for inflammation in the paws to monitor disease progression. b Clinical scores over time of the different treatment groups. c Changes in clinical scores starting from the day treatment was initiated in the different treatment groups. d Number of affected paws per treatment starting from the day treatment was initiated. e Number of affected paws with a clinical score of 5 or higher (considered to be severely affected paws) per treatment starting from the day treatment was initiated. f Clinical scores of paws that showed signs of arthritis at the start of treatment and extending over time, per treatment. g Clinical scores of paws that did not show signs of arthritis at the start of treatment, per treatment, over time. Values are mean ± SEM (n=11 per treatment group). Statistical analysis was performed using Student’s t test. *P < 0.05 abatacept + CD4 depletion vs CD4 depletion. CFA complete Freund’s adjuvant, IFA incomplete Freund’s adjuvant, PBS phosphate-buffered saline

Fig. 2

Incomplete CD4⁺ T cell depletion by GK1.5 over time. The presence of CD4⁺ T cells in the blood was monitored over time using flow cytometry. Blood was collected by making tail incisions during the experiment or by cardiac puncture at the of sacrifice. After red blood cell lysis, blood mononuclear cells were cell-surface stained for CD45, CD3, CD4 and CD8. Cells were gated on CD45 and subsequently on CD3 and CD4. a Dot plots of representative mice of each treatment group on day 12 and day 53 (end of experiment) after the start of treatment. b Summary of the percentages of CD4⁺ T cells per treatment at the end of follow-up is depicted for the blood, spleen and inguinal lymph node (LN). Each symbol represents 1 mouse. c Summary of CD4⁺ T cells in the blood over time as a percentage of the phosphate-buffered saline (PBS)-treated group. Values are mean ± SEM (n=11 per treatment group). Statistical analysis was performed using Student’s t test. *P < 0.05 abatacept + CD4 depletion vs CD4 depletion. ns not significant

Fig. 3

Abatacept decreased disease activity in thymectomized mice depleted of CD4⁺ T cells. a Collagen-induced arthritis was induced in male DBA/1 mice 2 weeks after they were thymectomized. When 80 % of the mice showed signs of arthritis, treatment was started. One day before the start of treatment, CD4⁺ T cells were depleted by intraperitoneal administration of GK1.5 and then the depletion was continued until the end of follow-up. Treatment was administered by intraperitoneal injection of PBS (diamonds), GK1.5 (squares; CD4 depl) or the combination of GK1.5 and abatacept (triangles; Aba + CD4 depl). The mice were scored three times per week for inflammation in the paws to monitor disease progression. b Clinical scores and changes in clinical scores starting from the day treatment was initiated in the different treatment groups (experiment 1; n=10 per treatment group). The same experiment was independently repeated in another 10 mice per treatment group, and an isotype for abatacept was used as control treatment (diamonds; iso) and in combination with CD4 depletion (cross symbols; iso + CD4 depl) (experiment 2). Changes in clinical scores starting from the day treatment was initiated are depicted. c The clinical scores of the paws that did not show signs of arthritis at the start of treatment are depicted for experiments 1 and 2. d The frequency of CD4⁺ T cells in blood at the end of follow-up was determined by flow cytometry. Abatacept-only treatment is not depicted to improve the readability of the graphs. Values are mean ± SEM. Statistical analysis was performed using Student’s t test. *P < 0.05 abatacept + CD4 depletion vs control group. CFA complete Freund’s adjuvant, IFA incomplete Freund’s adjuvant, ns not significant, PBS phosphate-buffered saline

Fig. 4

Reduced antibody levels after treatment with abatacept in the absence of CD4⁺ T cells. Antibody levels were determined by enzyme-linked immunosorbent assay over time. Serum samples were collected on days 0, 12, 21, 22, 35 and 47/48 after the start of treatment, and total IgG2a and IgG1 levels were determined in the thymectomized mice. In addition, bovine and mouse collagen type II–specific IgG2a and IgG1 levels were determined. a Levels are depicted as percentages of day 0. b An independent experiment including the isotype for abatacept. Abatacept-only treatment is not depicted to improve the readability of the graphs, but it showed results comparable to those for abatacept + CD4 depletion. Levels are depicted as percentages of day 0. Values are mean ± SEM (n=10 per treatment group). Statistical analysis was performed using the Mann–Whitney U test. *P < 0.05 abatacept + CD4 depletion vs control group. AU arbitrary units, bCII bovine collagen type II, CII collagen type II, IgG immunoglobulin G, mCII mouse collagen type II, PBS phosphate-buffered saline

Fig. 5

Abatacept decreased antibody levels detected in supernatants of ex vivo spleen and bone marrow cells in the absence of CD4⁺ T cells. After the animals were sacrificed, spleen and bone marrow cells were collected and cultured without stimulation for 7 or 14 days and then IgG production was measured in the supernatants by enzyme-linked immunosorbent assay. a Summary of the IgG levels detected in supernatants of spleen cells collected in experiments 1 and 2. The IgG levels of the phosphate-buffered saline (PBS)- and isotype-treated mice are combined and depicted as ‘PBS’, and the CD4 depletion and combination of isotype- and CD4 depletion–treated groups are combined and depicted as ‘CD4 depletion’. b Summary of the IgG levels detected in supernatants of bone marrow cells cultured for 7 or 14 days in experiments 1 and 2. Statistical analysis was performed using the Mann–Whitney U test. *P < 0.05 abatacept + CD4 depletion vs control group. AU arbitrary units, IgG immunoglobulin G