Cardiac xenotransplantation: re-emerged B cells after transient depletion by rituxan treatment maintains their repertoire but have a muted immune response
Avneesh Singh1, Philip Corcoran1, Billeta Lewis2, Marvin Thomas2, David Ayares3, Keith Horvath1, Muhammad Mohiuddin1.
1Cardiothoracic Surgery Research Program, NHLBI /NIH, Bethesda, MD, United States; 2DvR / ORS, NIH, Bethesda, MD, United States; 3Revivicor, Inc, Blacksburg, VA, United States
Background: Xenotransplantation (XTx) remains an option for end stage organ failure. Recently we have reported cardiac xenograft survival of over two year in pig to baboon heterotopic transplantation model. One major component of the modified immunosuppression (IS) is depletion of B cells by Rituxan (anti-CD20 antibody). In this study we have investigated the effect of Rituxan treatment on the recipient baboon and also evaluated the repertoire and function of re-emerging (RE) B cells.
Methods: Heterotopic cardiac XTx was performed from GTKOhCD46Tg (n=8) and GTKOhCD46TBM (n=5) pigs to baboons using an IS regimen that included anti thymocyte globulin, Rituxan on days -7, 0, 7, 14, mycophenolate mofitel, cobra venom factor, and co-stimulation blockade (anti-CD154 or anti-CD40) antibody. Baboon peripheral blood lymphocytes (PBLs) were collected twice a week for the first two months after cardiac XTx and then monthly thereafter. FACS analysis was done on PBLs with fluorescence conjugated anti-human CD3, CD4, CD5, CD19, CD20, CD24, CD38, IgD and IgM monoclonal antibodies to analyze the percentages of the B cells repertoire (plasma, naive, transitional and memory). Antibody secretion was also measured by ELISA from the supernatant of stimulated PBLs with anti IgM antibody.
Results: Heterotopic cardiac XTx was successfully achieved in recipient baboons and genetically modified cardiac xenograft survived up to 945 days with immunosuppression. Recipient baboons tolerated IS very well and Rituxan treatment depleted B cells completely at the time of XTx. However, B cells re-emerged after 8-10 weeks of the last dose of Rituxan. There was no significant difference found in relative percentages of RE- naive (CD20+CD24loCD38lo), transitional (CD20+CD24++CD38++) and memory (CD20+ CD27+) B cells as compared to naive animals. However, plasma cells (CD19loCD20neg and CD38++) were slightly increased in RE- B cells. We have also found that antibody secretion (IgG and IgM) by RE- B cells of long term XTx surviving baboons was significantly lower compared to the naive baboon.
Conclusion: Four doses of Rituxan treatment in cardiac XTx recipients deplete B cells completely for about 2 months, and after 8-10 weeks B cells (RE-B cells) return to a normal repertoire. With recovery after transient B cell depletion antibody secretion from RE- B cells was significantly reduced in baboons with long term surviving xenograft.
NHLBI Animal Surgery and Resources. DVR/ ORS/ NIH.
11:00 - 12:30
|Solid Organ Xenotransplantation (2)||Cardiac xenotransplantation: Role of CD4+CD25hiFoxP3+ T regulatory cells in long-term cardiac xenograft survivors||Room 109|
15:30 - 17:00
|Solid Organ Xenotransplantation (1)||Cardiac xenotransplantation: re-emerged B cells after transient depletion by rituxan treatment maintains their repertoire but have a muted immune response||Room 109|