Anti-CD28 Antibody-Initiated Cytokine Storm in Canines.

BACKGROUND: CD28 signal blockade following T cell receptor activation is under intense investigation as a tolerance-inducing therapy for transplantation. Our goal is to produce a CD28-specific reagent as a therapy for the prevention of graft rejection and graft-versus-host disease in the canine model of allogeneic hematopoietic cell transplantation (HCT). METHODS: We infused a monoclonal mouse anti-canine CD28 antibody (1C6 mAb) into four dogs and a fragment of antigen-binding (1C6 Fab) into two dogs. Pharmacokinetics, pathology, cytokine release, and the crystal structure of 1C6 Fv were evaluated. RESULTS: Within an hour of an IV injection of the 1C6 mAb, the dogs became leukopenic and developed a steroid-refractory cytokine storm. Two of the dogs developed high fevers, one experienced diffuse alveolar hemorrhage, and another developed gastrointestinal hemorrhage. The cytokine storm was characterized by elevated plasma levels of MCP-1, IP-10, IL-10, IL-6, and TNF-α. In addition, one dog showed elevated levels of IL-2, IL-8, and IL-18. In contrast, infusion of 1C6 Fab was well tolerated without any side effects. Dry-coating 1C6 mAb onto tissue culture plates induced CD3-independent proliferation and TNF-alpha production. Crystal structure analysis revealed that 1C6 binds to canine CD28 in a manner different than previously reported for conventional agonistic or superagonistic antibodies. CONCLUSIONS: These results indicate that dogs and humans develop a similar cytokine storm following infusion ofanti-CD28 mAb, providing an appropriate large animal for further study. 1C6 Fab warrants evaluation as a tolerance-inducing reagent in the canine model of allogeneic HCT.

A novel monoclonal antibody specific for canine CD25 (P4A10): selection and evaluation of canine Tregs.

A monoclonal antibody (mAb), P4A10, was made to the canine interleukin-2 receptor alpha chain (IL-2Ralpha; p55; Tac antigen; CD25) to facilitate studies of canine regulatory T-cells (Treg). By non-reduced Western blot, P4A10 bound to a 55kDa protein, the size of human IL-2Ralpha. In flow cytometry assays, it reacted with a minor population of circulating dog CD3(+)CD4(+) T-cells and the majority (>60%) of in vitro PMA-Ionomycin (PMA-IO)-activated canine CD3(+) T-cells. P4A10 recognized a hematopoietic cell population enriched for FoxP3+ cells as measured by flow cytometry. The P4A10-selected fractions of T-cells had significantly increased copy numbers of CD25, FoxP3, IL-10, and TGFbeta as detected by RT-PCR (reverse transcriptase-PCR) compared to the negative fractions. The P4A10-selected cells inhibited (3)H (tritiated) thymidine incorporation in a mixed leukocyte reaction (MLR) containing responders of the same origin. P4A10-selected T-cells from fresh peripheral blood mononuclear cells had less FoxP3 (p=0.07) by qRT-PCR (quantitative RT-PCR) and were less suppressive (p=0.01) than in vitro alloantigen-activated Treg. The mAb P4A10 is specific for canine CD25 and can be used to facilitate studies of CD25+FoxP3+ Treg in this clinically relevant large animal model.

Evaluation of different methods of leukoreduction of donor platelets to prevent alloimmune platelet refractoriness and induce tolerance in a canine transfusion model.

The effectiveness of different methods of leukoreduction in preventing alloimmune platelet refractoriness was evaluated in a canine model. Platelets from a random donor dog were administered for up to 8 weeks or until platelet refractoriness. Standard (STD; unmodified) platelets were accepted by 14% of recipients (n = 7) compared with 14% for centrifuge leukoreduced (C-LR) platelets (n = 21) and 31% for filter leukoreduced (F-LR) platelets (n = 13; no significant differences). Surprisingly, using both F-LR and C-LR platelets was highly effective (87% acceptance, n = 15). Transfusing F-LR/C-LR red blood cells (n = 4) or F-LR/C-LR plasma (n = 4), along with F-LR/C-LR platelets, did not affect platelet acceptance (100% acceptance). Overall acceptance of F-LR/C-LR platelets was 91% (n = 23; P < or = .05 versus STD, C-LR, or F-LR platelets). F-LR/C-LR transfusions also induced tolerance to subsequent STD platelet transfusions from the same donor (82% acceptance, n = 19) as well as to donor skin grafts without recipient immunosuppression (57% acceptance, n = 7). To evaluate mechanisms of tolerance induction, F-LR/C-LR platelets were gamma-irradiated. Although the gamma-irradiated F-LR/C-LR platelets were uniformly accepted (n = 6), tolerance to STD platelets was lost. These data suggest that some allostimulatory white cells are filter adherent, whereas others escape filtration but can be removed by centrifugation and tolerance requires a residual functioning white cell.