K. Onodera

The effects of nondepleting CD4 targeted therapy in presensitized rat recipients of cardiac allografts

The immunosuppressive effects of RIB-5/2, a nondepleting anti-rat CD4 monoclonal antibody (mAb), were analyzed in a well-defined model of accelerated cardiac allograft rejection. (LEW x BN)F1 hearts are rejected within 24 hours in LEW hosts presensitized with BN skin grafts at day -7. Treatment with RIB-5/2 mAb (3.5 mg/day i.v.) at days -7 and -1, prolonged cardiac allograft survival to the median of >62 days. The long-term recipients rejected acutely third-party (Wistar-Furth) test skin grafts, without an adverse effect on the survival of the original cardiac transplants. Lymphocytes harvested from mAb-treated hosts significantly decreased proliferative responses of donor cells in mixed leukocyte reaction. The cell activation and cytokine elaboration patterns were evaluated at the mRNA and protein levels by competitive template reverse transcriptase polymerase chain reaction and immunohistochemistry, respectively. Cardiac allografts in CD4 mAb-treated rats at 24 hours displayed reduced CD3, CD25, tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-2, interferon (IFN)-gamma, and IL-10 mRNA levels as compared to those in rejecting grafts. Equal amounts of IL-4 mRNA were detected throughout in both animal groups; the expression of IL-10 mRNA increased progressively in the treated hosts. In contrast, IFN-gamma was consistently depressed after mAb therapy. The mRNA levels coding for CD3, CD25, tumor necrosis factor-alpha, IL-1-beta, and IL-2 genes were comparable in long-surviving and rejecting allografts. The staining for IL-2R, IL-2, and IFN-gamma was diminished, whereas the staining for IL-4 was either unaffected or enhanced in well-functioning grafts in RIB-5/2 mAb-treated hosts. The untreated recipients elicited strong circulating IgM allo-Ab response, which peaked around the time of cardiac rejection and then switched to IgG allo-Ab 4-7 days after heart transplantation. Treatment with RIB-5/2 mAb decreased IgM and prevented the switch into the IgG allo-Ab response. In conclusion, the ability of RIB-5/2 mAb treatment to combat accelerated rejection and to produce long-term graft acceptance is unprecedented in our experience in this model. These data provide new insights into the complexities of the cellular and humoral responsiveness, contributing to the the induction of donor-specific unresponsiveness in sensitized hosts. This study, along with our previous reports, indicate that an immune deviation in which intragraft Th1-type cytokines (primarily IFN-gamma) are diminished and Th2-type cytokines (IL-4 and IL-10) are maintained represents the common effector mechanism of CD4 mAb regimens in recipients of vascularized organ allografts.

Distinct tolerance pathways in sensitized allograft recipients after selective blockade of activation signal 1 or signal 2.

BACKGROUND CD4-targeted therapy or blocking of CD28-B7 T-cell costimulation may produce indefinite cardiac allograft survival in presensitized rats. This study analyzes the immune events associated with tolerance pathways after the blockade of activation signal 1 (CD4 monoclonal antibody [mAb]) or signal 2 (CTLA4Ig). METHODS AND RESULTS Lewis rats sensitized with Brown Norway skin grafts reject LBNF1 cardiac allografts in <36 hr. Animals were treated with RIB-5/2, a nondepleting CD4 mAb, or with CTLA4Ig + LBNF1 spleen cells. RIB-5/2 monotherapy uniformly produced permanent cardiac graft acceptance, whereas CTLA4Ig produced indefinite graft survival in about 50% of sensitized rats. Spleen cells (100 x 10(6)) from CD4 mAb-treated rats conferred donor-specific tolerance after transfer into new sets of recipients. This tolerant state could be then transferred with regulatory cells in an infectious manner into new cohorts of engrafted rats. In contrast, features of infectious tolerance could be detected in CTLA4Ig-treated hosts after infusion of >300 x 10(6) of splenocytes. CD4 mAb therapy abolished the transcription of both T helper (Th)1 and Th2 cytokines compared with rejecting controls. In contrast, CTLA4Ig treatment resulted in a selective sparing of Th2-type cytokines. Surviving grafts in both groups were largely protected from signs of chronic rejection. CONCLUSIONS CD4 mAb-induced blockage of activation signal 1 or CTLA4Ig-mediated blockage of costimulatory signal 2 may induce a true transplantation tolerance in sensitized rats, as documented by permanent graft acceptance and attenuation of chronic injury. The infectious pathway operates in a cell dose-dependent manner. Th2-type deviation in the graft itself is not required for tolerance maintenance, and it does not necessarily lead to chronic injury.