High-Dose Porcine Hematopoietic Cell Transplantation Combined With Cd40 Ligand Blockade in Baboons Prevents an Induced Anti-Pig Humoral Response

The majority of xenoreactive natural Abs in humans recognize the carbohydrate Ag present on pig tissue, Galα1–3Galβ1–4GlcNAc-R (αGal), synthesized by the enzyme UDP galactose:β-d-galactosyl-1,4-N-acetyl-d-glucosaminide α(1–3)galactosyltransferase or αGT. Using αGT knockout mice (GT0 mice), which like humans produce serum Abs that bind αGal, we examined the role of T cells in production of Abs specific for αGal. GT0 mice were crossed with TCR-β knockout mice (TCR-β0) to generate double-knockout mice (GT0/TCR-β0). While GT0/TCR-β+ mice exhibited an age-dependent increase in the serum titer of natural Abs specific for αGal, a similar increase was not observed in GT0/TCR-β0 mice, and the titer of αGal-specific Abs in double knockouts was significantly lower than in age-matched GT0/TCR-β+ mice. Immunization with pig cells resulted in a significant increase in the serum titer of αGal-specific Abs in GT0/TCR-β+ mice, but had no effect on the level of αGal-specific serum Abs in GT0/TCR-β0 mice. Treatment of GT0/TCR-β+ mice with anti-CD40L Abs before immunization with pig cells prevented sensitization to αGal. Our data suggest that the majority of αGal-specific Abs are T cell dependent and that production of αGal-specific Abs after sensitization can be prevented by blocking costimulatory pathways.

Section: Discussionmentioning

confidence: 77%

The Role of T Cell Help in the Production of Antibodies Specific for Galα1–3Gal

Crétin

Bracy

Hanson

et al. 2002

“…It is clear that two different populations of B cells contribute to the anti-Gal Ab response. The first is a Tindependent B-1B cell subset that is responsible for T-independent anti-Gal IgM production (32), and the second is a conventional T-dependent B-2 population that produces anti-Gal IgG (17,(33)(34)(35). It has recently been reported that the signaling thresholds for B-1 and B-2 cells differ, and differentiation of B-0 and B-2 cells to B-1 cells that have increased activation thresholds can result in functional B cell tolerance (36 -38).…”

Section: Discussionmentioning

confidence: 99%

Intact Active Bone Transplantation Synergizes with Anti-CD40 Ligand Therapy to Induce B Cell Tolerance

Yin

Varghese

et al. 2002

Blockade of T cell costimulatory pathways can result in the prolongation of allograft survival through the suppression of Th1 responses; however, late allograft rejection is usually accompanied by an emerging allograft-specific humoral response. We have recently determined that intact active bone (IAB) fragments transplanted under the kidney capsule can synergize with transient anti-CD40 ligand (CD40L) treatment to induce robust donor-specific allograft tolerance and suppress the alloantibody response. In this study, we take advantage of the ability of galactosyltransferase-deficient knockout (GT-Ko) mice to respond to the carbohydrate epitope, galactose-α1,3-galactose (Gal), to investigate whether IAB plus transient anti-CD40L therapy directly tolerize B cell responses. GT-Ko mice tolerized to Gal-expressing C3H hearts and IAB plus transient anti-CD40L therapy were challenged with pig kidney membranes that express high levels of Gal. The anti-Gal IgM and IgG responses were significantly suppressed in IAB-tolerant mice compared with controls, while the non-Gal anti-pig Ab responses were comparable. The anti-pig T cell cytokine response (IFN-γ and IL-4) was comparable in IAB-tolerant and control mice. The tolerant state for the anti-Gal IgM response could be reversed with repeated immunization, whereas the tolerant state for the IgG response was robust and resisted repeated immunization. These observations provide an important proof-of-concept that adjunct therapies can synergize with anti-CD40L Abs to tolerize B cell responses independent of their effects on T cells. This model, which does not require mixed chimerism, provides a unique opportunity for investigating the mechanism of peripheral tolerance in a clinically relevant population of carbohydrate-specific B cells.

“…The R/S ratio represents the numbers of replacement/silent mutations as previously described (20). The relative differences in affinity between hybridomas (listed in affinity measurements and suggest that upon affinity maturation some anti-Gal IgG from Gal Ϫ/Ϫ mice can attain affinity comparable to that of humans or primates.…”

Section: Affinity Of Anti-gal Hybridoma Absmentioning

confidence: 99%

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

Yin

Naziruddin

et al. 2003

We have previously produced a series of antigalactose (anti-Gal) hybridomas and characterized their heavy chain gene usage. Here we have quantified the affinity of these Abs for the α-Gal epitope and characterized their in vitro effects on endothelial cell activation and apoptosis. We report that anti-Gal mAbs derived from Gal−/− mice show a range of affinity for the α-Gal epitope, and that affinity was generally increased as the VH gene usage transitioned from germline sequences to sequences exhibiting somatic maturation. Despite an 85-fold range in affinity, all the anti-Gal mAbs examined induced α-Gal-specific endothelial cell activation, and after prolonged exposure induced endothelial cell apoptosis in a complement-independent manner. Only murine anti-Gal mAbs of the IgM or IgG3 subclass, but not IgG1, were effective at initiating complement-dependent cell lysis. Using a novel rat to mouse xenograft model, we examined the in vivo ability of these mAbs to induce xenograft rejection and characterized the rejection using histology and immunohistochemistry. Infusion of complement-fixing IgG3 mAbs resulted in either hyperacute rejection or acute vascular rejection of the xenograft. Surprisingly, infusion of an equal amount of a high affinity anti-Gal IgG1 mAb, that fixed complement poorly also induced a rapid xenograft rejection, which we have labeled very acute rejection. These studies emphasize the importance of in vivo assays, in addition to in vitro assays, in understanding the role of anti-Gal IgG-mediated tissue injury and xenograft rejection.