Shengqiao Li, Yuan Lin, Yehong Yan, Omer Rutgeerts, Caroline Lenaerts, An D Billiau, Mark Waer

1. Absence of Xeno-rejection and Xenoantibody Production in IL-7 KO Recipients After Xenoheart, but not after Xenoskin Transplantation. Shengqiao Li, Yuan Lin, Yehong Yan, Omer Rutgeerts, Caroline Lenaerts, An D Billiau, Mark Waer Laboratory of Experimental Transplantation University of Leuven Belgium

2. Introduction • IL-7 is a critical cytokine for T and B cell survival and development in mice. It was first discovered in 1988 as a factor that promoted the growth of murine B cell precursors in a bone marrow culture system. • IL-7 is produced by the non-lymphoid cells in lymphoid organs. It is a member of the gamma chain-dependent family of cytokines, including IL-2, IL-7, IL-9, and IL-15. • IL-7 KO mice • exhibit impaired T cell development with a 10- to 100-fold reduction in the number of mature T cells in secondary lymphoid organs . In vivo administration of IL-7 was shown to increases basal proliferation in CD4 and CD8 T cells • in the B cell lineage, IL-7KO mice show a reduction of splenic B cells and an abnormal population of immature B cells the residual splenic T and B cells show normal responsiveness to mitogeneic stimuli. • IL-7-KO exhibit no effect on NK cells and macrophages. Carvalho TL et al. Arrested B lymphopoiesis and persistence of activated B cells in adult interleukin 7(-/)- mice. J Exp Med. 2001, 194(8):1141-50. Tan JT et al. IL-7 is critical for homeostatic proliferation and survival of naive T cells. Proc Natl Acad Sci U S A 2001, 98(15):8732-7.

3. Aims • To investigate whether or not deficiency of IL-7 results into long-term survival of xenografts. • To investigate the mechanisms involved.

4. Results: phenotypical analysis of lymphocytes in IL-7 KO mice Conclusions:1. IL-7 KO mice exhibit a severe reduction of absolute lymphocyte numbers in the spleen, which are 5-10 times lower than IL-7 WT mice.2. Both T and B cells are reduced, while NK cell numbers are not affected.3. As for the B cell subpopulation, MZ B cells are less affected than FO B cells.

5. Results: xenoheart survival in IL-7 KO and WT mice Naive heart D100 in IL-7 KO D6 in IL-7 WT Conclusion:1. Xeno-hearts have a long-term survival (>120 days) in IL-7 KO mice. By contrast, IL-7 WT mice reject xeno-hearts as fast as C57BL/6 euthymic mice.2. As T deficient nude mice are able to reject xeno-hearts, the mechanism involved in the failure of IL-7 KO mice to reject xenohearts must involve reasons other than T cell deficiency3. In long-term surviving xenografts, neointima proliferation is found, indicating that IL-7 deficiency does not prevent chronic rejection.

6. Results: anti-hamster IgM Xab formation after xenoheart transplantation Conclusion :IL-7 KO mice fail to produce high titers of IgM anti-hamster xeno-antibody.

7. Results: survival of xeno-skin grafts in IL-7 KO mice, and IgM/IgG xenoantibody productions Conclusion:Xeno-skins are rejected after 2-4 months, and associated with delayed IgG Xab production

8. Results: adoptive lymphocyte transfer at day of heart transplantation in IL-7 KO mice Conclusion :1. IL-7 KO mice are able to reject xeno-hearts after transfer of naive or presensitized lymphocytes from WT euthymic mice at the day of transplantation. However, they did not reject xenoheart after transfer of lymphocytes from IL-7 KO mice.2. This observation indicates that xenoheart rejection can be triggered in the absence of IL-7.

9. Results: adoptive lymphocyte transfer at day 3 to day 10 before xenoheart transplantation in IL-7 KO mice Conclusion:Lymphocytes from WT euthymic mice and not from IL-7KO mice are able to induce xenoheart rejection in an IL-7 KO environment, even when administered 10 days before transplantation (which may be expected to impair this function when depending on IL-7)

10. Results: NK xeno-cytotoxicity in IL-7 KO mice Conclusion:Functional NK xeno-cytotoxicity is normal in IL-7 KO mice

11. Conclusions • Despite normal NK cell numbers and function, and despite reasonable MZ B cell numbers (previously shown by us to be both essential for rapidly induced IgM Xab production), T-independent IgM Xab production is deficient in IL-7KO mice, resulting in absence of acute cardiac xenograft rejection. This suggests that qualitative defects in MZB cells or defects in a third factor (other than MZ B and NK cells) are involved in defective IgM Xab induction • The low number of T cells in IL-7KO mice probably explains: on the one hand, the occurence of chronic vascular lesions in long-term xenoheart recipients on the other hand, the delay in xenoskin rejection • The effects of IL-7 absence are probably related to defects in the generation and/or function of several lymphocyte subsets and can be corrected by administration of normal splenocytes, suggesting that acute blockade of IL-7 will not be effective to influence xenograft rejection

12. Materials and Methods • Animals: Recipient mice: 1. IL-7 KO mice (C57BL/6 background) were kindly provided by P Vieira (Paris, France) 2. C57BL/6 euthymic mice, purchased from Harlan (Netherlands). Xenogeneic donor animals: 1. Inbred golden Syrian hamster, bred locally (University of Leuven). 2. Inbred 30-100g RA rats, bred locally (University of Leuven) • Flow cytometric analysis: 1. Phenotypical analysis IL-7KO mice: Anti-mouse CD3, CD4,CD8, B220, CD21,CD23, DX5,NK1.1 antibodies ( FITC or PE or Per-cp) purchased from BD Biosciences, Belgium marginal zone (MZ) B cells are gated as B220+CD21 high CD23low, follicular cells are gated as B220+CD21lowCD23high. 2. Measurement of IgM and IgG xenoantibodies: Anti-mouse IgM and IgG (FITC), purchased from SeroTec • Adoptive transfer experiments: Splenocytes (+/-lysis of RBC) were injected into the tail vein of recipients. • NK xenocytotoxicity: NK cells were isolated by DX5 microbeads to be used for NK cytotoxicity.