Non‐depleting anti‐CD4, but not anti‐CD8, antibody induces long‐term survival of xenogeneic and allogeneic hearts in α1,3‐galactosyltransferase knockout
(GT‐Ko) mice

Abstract

The anti-galactose-alpha1,3-galactose (Gal) antibody (Ab) response following pig-to-human transplantation is vigorous and largely resistant to currently available immunosuppression. The recent generation of GT-Ko mice provides a unique opportunity to study the immunological basis of xenograft-elicited anti-Gal Ab response in vivo, and to test the efficacy of various strategies at controlling this Ab response [1]. In this study, we compared the ability of non-depleting anti-CD4 and anti-CD8 to control rejection and antibody production in GT-Ko mice following xenograft and allograft transplantation. Hearts from baby Lewis rat or C3H mice were transplanted heterotopically into GT-Ko. Non-depleting anti-CD4 (YTS177) and anti-CD8 (YTS105) Abs were used at 1 mg/mouse, and given as four doses daily from day -2 to 1 then q.o.d. till day 21. Xenograft rejection occurred at 3 to 5 days post-transplantation in untreated GT-Ko recipients, and was histologically characterized as vascular rejection. Anti-CD4, but not anti-CD8, Ab treatment prolonged xenograft survival to 68 to 74 days and inhibited anti-Gal Ab as well as xeno-Ab production. In four of the five hearts from anti-CD4 mAbs-treated GT-Ko mice, we observed classic signs of chronic rejection, namely, thickened intima in the lumen of vessels, significant IgM deposition, fibrosis and modest mononuclear cell infiltrate of Mac-1+ macrophages and scattered T cells (CD8>CD4). Xenograft rejection in untreated, as well as anti-CD4- and anti-CD8-treated, recipients was associated with increased intragraft IL-6, IFN-gamma and IL-10 mRNA. C3H allografts were rejected in 7 to 9 days by untreated GT-Ko mice and were histologically characterized as cellular rejection. Treatment with anti-CD4 and anti-CD8 mAb resulted in graft survivals of >94.8 and 11.8 days, respectively. Anti-CD4 mAb treatment resulted in a transient inhibition of alloreactive and anti-Gal Ab production. The presence of circulating alloreactive and anti-Gal Abs at >50 days post-transplant was associated with significant IgM and IgG deposition in the graft. Yet, in the anti-CD4 mAb-treated group, the allografts showed no signs of rejection at the time of sacrifice (>100 days post-transplantation). All rejected allografts had elevated levels of intragraft IL-6, IFN-gamma and IL-10 mRNA, while the long-surviving anti-CD4-treated allografts had reduced mRNA levels of these cytokines. Collectively, our studies suggest that the elicited xeno-antibody production and anti-Gal Ab production in GT-Ko mice are CD4+ T-cell dependent. The majority of xenografts succumbed to chronic rejection, while allografts survived with minimal histological change, despite elevated levels of circulating alloAbs. Thus, immunosuppression with anti-CD4 mAb therapy induces long-term survival of allografts more effectively than to xenografts.

The In Vitro and In Vivo Effects of Anti-Galactose Antibodies on Endothelial Cell Activation and Xenograft Rejection

“…Heterotopic rat hearts were transplanted into the abdomen of the recipient by anastomosing the donor aorta to recipient aorta and the donor pulmonary artery to recipient inferior vena cava as previously described (33). Anti-Gal mAb were administered by i.v.…”

Acute Xenograft Rejection Mediated by Antibodies Produced Independently of TH1/TH2 Cytokine Profiles

“…The kinetics were delayed and the titers of the antigraft IgG response was significantly reduced in C57BL/6 compared with BALB/c recipients. We have previously reported that the mouse antirat IgM response is T-cell dependent and is inhibited by nondepleting anti-CD4 mAbs or costimulation blockade with CTLA4-Ig and anti-154 (22,25). However neither the rate nor magnitude of the antigraft IgM response was affected by the absence of IFN-g or IL-4.…”

“…Lewis rats were purchased from Harlan (Walkersville, MD). Mice received heterotopic cardiac transplants from allo (BALB/c) or xeno (Lewis) donors, as previously described (21,22). The heart grafts were monitored daily until rejection, and rejection was defined as complete cessation of pulsation.…”

“…Alloreactive IgM, IgG and IgG subclasses in the serum samples were quantitated by flow cytometry as previously described, using rat erythrocytes, BALB/c or C57BL/6 lymph node cells as targets (22 cells were incubated at 37 aeC for 72 h. The supernatant was collected and kept at ª70 aeC. The IL-4 and IFN-g concentrations of the supernatants were determined using the ELISA OptEIA kits.…”

Hyperacute Rejection by Anti-Gal IgG1, IgG2a, and IgG2b Is Dependent on Complement and Fc-γ Receptors

“…The ␣1,3-galactosyltransferase (Gal Ϫ/Ϫ ) knockout mouse, which lacks the expression of the Gal epitope, has been used as a recipient for studying anti-Gal responses to Gal ϩ/ϩ cardiac xenografts (12)(13)(14)(15)(16). Preformed anti-Gal Abs are primarily IgM, whereas the elicited anti-Gal IgG Abs following xenotransplantation of Lewis rat hearts were restricted to the IgG3 and IgG1 subclasses (14).…”