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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

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Shengqiao li yuan lin yehong yan omer rutgeerts caroline lenaerts an d billiau mark waer

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


Introduction

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.


Shengqiao li yuan lin yehong yan omer rutgeerts caroline lenaerts an d billiau mark waer

Aims

  • To investigate whether or not deficiency of IL-7 results into long-term survival of xenografts.

  • To investigate the mechanisms involved.


Shengqiao li yuan lin yehong yan omer rutgeerts caroline lenaerts an d billiau mark waer

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.


Results xenoheart survival in il 7 ko and wt mice

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.


Results anti hamster igm xab formation after xenoheart transplantation

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.


Results survival of xeno skin grafts in il 7 ko mice and igm igg xenoantibody productions

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


Results adoptive lymphocyte transfer at day of heart transplantation in il 7 ko mice

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.


Shengqiao li yuan lin yehong yan omer rutgeerts caroline lenaerts an d billiau mark waer

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)


Results nk xeno cytotoxicity in il 7 ko mice

Results: NK xeno-cytotoxicity in IL-7 KO mice

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


Conclusions

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


Materials and methods

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.


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