Ctla4 ig

Bilateral dorsal full-thickness tail and ear skin were transplanted onto recipient mice. Skin graft recipients received no treatment, anti-CD28 domain antibody (dAb) (100 μg, Bristol-Myers Squibb), CTLA-4-Ig (200 μg, Bristol-Myers Squibb), or anti-CTLA-4 mAb (9H10, 250 μg, BioXcell). Treatments were administered intraperitoneally on post-transplant days 0, 2, 4, 6 and 8, and then weekly thereafter. Anti-CD28 dAb and CTLA-4-Ig dosing was based on molecular weight, serum half-life, and murine mixed lymphocyte reaction EC₅₀ (14 (link), 20 (link)).

Heterotopic heart transplantations were performed as previously described [31 (link)] by grafting BALB/c hearts from 6-8 week old donors onto the aorta and inferior vena cava in the peritoneal cavity of 8-12 week old C57BL/6 recipients. 500 μg of CTLA4-Ig (abatacept; Bristol-Myers Squibb) per mouse, intraperitoneally, starting on day −2, 0 and 2 or on days 6, 8, 10, and then twice per week until the end of the experiment.

For adoptive transfers of OVA-specific T cells, splenocytes isolated from Thy1.1⁺ OT-I and Thy1.1⁺ OT-II mice were quantified via TruCount bead analysis (BD Biosciences) and 1.0×10⁶ of each Thy1.1⁺ OT-I and Thy1.1⁺ OT-II T cells was injected intravenously into naïve B6-Ly5.1/Cr mice 24–48 hours prior to skin transplantation. Bilateral dorsal full-thickness tail and ear skin were transplanted onto recipient mice (23 (link)). Skin graft recipients received no treatment, anti-CD28 domain antibody (dAb) (100 μg, Bristol-Myers Squibb), CTLA-4-Ig (200 μg, Bristol-Myers Squibb), or tacrolimus (5 mg/kg, Astellas). Anti-CD28 dAb and CTLA-4-Ig were administered intraperitoneally on post-transplant days 0, 2, 4, 6 and 8 and then weekly. Delayed anti-CD28 dAb was administered on days 7, 9, 11, 13 and 15 and then weekly. Tacrolimus was administered s.c. daily with mean trough levels of 7.1 ± 3.2 ng/mL.

Animal studies were approved by the Animal Care and Use Committees of the Atlanta US Department of Veterans Affairs Medical Center and of Emory University.
Mice were housed under specific pathogen free conditions and fed gamma-irradiated 5V02 mouse chow (Purina Mills, St. Louis, MO) and autoclaved water ad libitum.
All studies involved C57BL6 female mice. Wild-type (WT), T cell receptor beta gene (Tcrb) knockout (KO) mice (deficient in αβ T cells; hereafter, TCRβ KO) were from Jackson Labs (Bar Harbor, ME). Wnt-10b gene (Wnt10b) KO mice (hereafter, Wnt-10b KO) were from an Emory breeding colony and have been previously described.^{24 (link)–26 (link)}Mice were injected intraperitoneally with 1 mg/Kg CTLA-4Ig (Orencia: Bristol-Myers Squibb) twice weekly, or with control human Ig (Lampire Biological Laboratories, Pipersville, PA) for 3 or 6 months as indicated.

Heterotopic heart transplantation was performed as previously described ³³ (IACUC #M019M352), n=5 surgery survival animals per group. Recipients were administrated with a single dose of CTLA4-Ig (Bristol-Myers Squibb, Princeton, NJ) on day of transplant (d0, 500μg intraperitoneal). The grafts were monitored by daily palpation and graded from 4+ (strong beat) to 0 (no beat) by two individuals in a blinded manner and confirmed by laparotomy at the time of sacrifice ³⁴ . Rejection was defined as beating score 1 (minimally palpable beat) or 0 (no palpable beat). The animals were sacrificed on the day of beating score 1 or at a designated time endpoint.
Due limitations imposed by the complex transgenic breeding strategy and surgical approach to obtain balanced cohorts, only male mice were included in the study. We acknowledge the limitations imposed by this design.
CTLA4-Ig has been widely described as a mean to achieve complete allogeneic tolerance in mice ^{35 (link), 36 (link)}, as well as immune regulation in humans ³⁷ . The latter is achieved via complex immunologic mechanisms, including CD80/86-CD28 axis blockade ^{38 (link), 39 (link)}. In this study we used a sub-therapeutic CTLA4-Ig scheme to generate partial/transient allo-tolerance in transplanted recipients, aiming to resemble the drug-induced partial tolerance of actual human patients enrolled in the study.