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Increased Soluble CD154 (CD40 Ligand) Levels in Xenograft Recipients Correlate With the Development of De Novo Anti-Pig IgG Antibodies

Ezzelarab, Mohamed B.1,5; Ekser, Burcin1,2; Isse, Kumiko1; Iwase, Hayato1; Morelli, Adrian E.1,3; Ayares, David4; Cooper, David K.C.1

Transplantation:
doi: 10.1097/TP.0000000000000042
Basic and Experimental Research
Abstract

Background: De novo anti-pig antibodies are associated with acute humoral xenograft rejection. We explored the relative efficacy of CD40/CD154-pathway blockade versus CD28/B7-pathway blockade in the prevention of de novo anti-pig IgG antibodies in xenograft recipients.

Methods: After α1,3-galactosyltransferase gene-knockout pig artery patch xenotransplantation, recipient baboons received no immunosuppression (IS; n=3), or anti-CD154mAb-based (n=5) or CTLA4-Ig-based (n=5) IS. CD4+ T-cell and CD20+ B-cell numbers in blood were determined. Serum anti-pig IgG antibodies and serum soluble (s)CD154 levels were measured. In lymph nodes, germinal center formation was examined and numbers of proliferating cells were evaluated by Ki-67 staining.

Results: After transplantation, with no IS, CD4+ T-cell and CD20+ B-cell numbers were increased, but were reduced by IS.

In lymph nodes, with no IS, there was enhanced germinal center formation, which was significantly reduced by anti-CD154mAb-based (P<0.01) or CTLA4-Ig-based (P<0.01) IS. With no IS, there was strong expression of Ki-67-positive cells in lymph nodes, indicating extensive cellular proliferation. Ki-67-positive cells were significantly reduced by anti-CD154mAb-based (P<0.05) but not by CTLA4-Ig-based IS. High mean levels of sCD154 were detected with no IS (3324 pg/mL), in comparison to naive control baboons (214 pg/mL). With anti-CD154mAb-based IS, sCD154 was reduced to less than 1 pg/mL and with CTLA4-Ig-based IS to 65 pg/mL. There was significant positive correlation between sCD154 and anti-pig IgG levels (P<0.01).

Conclusions: In xenograft recipients, anti-CD154mAb may reduce class-switching of anti-pig antibodies by binding both T-cell surface CD154 and circulating sCD154, thus preventing subsequent stimulation of B cells and activation of lymphoid follicles in secondary lymphoid tissues.

Author Information

1 Thomas E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh, Pittsburgh, PA.

2 Department of Surgery, Transplantation and Advanced Technologies, Vascular Surgery and Organ Transplant Unit, University Hospital of Catania, Catania, Italy.

3 Department of Immunology, University of Pittsburgh, Pittsburgh, PA.

4 Revivicor, Blacksburg, VA.

5 Address correspondence to: Mohamed B. Ezzelarab, M.D., Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, Starzl Biomedical Science Tower, E1551, 200 Lothrop Street, Pittsburgh, PA 15261, USA.

M.B.E. is a recipient of a Joseph A. Patrick Research Fellowship in Transplantation from the Thomas E. Starzl Transplantation Institute. B.E. is a recipient of NIH NIAID T32 AI 074490 Training Grant. Work in our laboratories is supported in part by NIH grants #U01 AI068642, #R21 A1074844, #U19 A1090959, P01 HL107152, and by Sponsored Research Agreements between the University of Pittsburgh and Revivicor, Inc.

D.A. is an employee of Revivicor, Inc., Blacksburg, VA. The other authors declare no conflicts of interest.

E-mail: ezzemb@upmc.edu

M.B.E. and D.K.C. participated in designing the study, performing the research, analyzing the data, and initiating the original draft of the article. K.I. participated in analyzing the data. B.E. and H.I. participated in performing the research. All authors contributed to the interpretation of the data and revising the draft to produce the final format of the article.

Supplemental digital content (SDC) is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.transplantjournal.com).

Received 12 August 2013. Revision requested 3 September 2013.

Accepted 28 November 2013.

Accepted January 31, 2014

© 2014 by Lippincott Williams & Wilkins