shengqiao li, yuan lin, yehong yan, omer rutgeerts, caroline lenaerts, an d billiau, mark waer
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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 - PowerPoint PPT PresentationTRANSCRIPT
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 TransplantationUniversity of Leuven
Belgium
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 cellsthe 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.
Aims
• To investigate whether or not deficiency of IL-7 results into long-term survival of xenografts.
• To investigate the mechanisms involved.
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.
tota
l spl
enoc
ytes
( x
106
)
0
20
40
60
80
100
IL-7-/- C57BL/6 euth
abso
lute
cou
nt in
spl
een
(x 1
06)
0
10
20
30
40
50
CD3DX5B220MZ BFO B
IL-7-/- C57BL/6 euthymic
Results: xenoheart survival in IL-7 KO and WT mice
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.
Survival hamster heart xenogreafts
days after transplantation
0 5 10 15 20 110 115 120
% s
urvi
val
0
20
40
60
80
100
IL-7 -/- (n=7)WT littermate (n=5)C57BL/6 nude (n=5)C57BL/6 euthymic (n=5)
Naiveheart
D100 in IL-7 KO
D6in IL-7 WT
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.
serum IgM Xab
IL-7 -/- naive IL-7 -/- Xgraft WT Xgraft C57Bl/6 eut Xgraft
mean flu
ore
scence inte
nsity
0
50
100
150
200
250
300
350
n=2 n=4 n=4 n=3
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
Survival rat skin xenografts
days after transplantation
0 10 20 30 40 50 60 70 80 90 100 110 120
% s
urvi
val
0
20
40
60
80
100
IL-7 -/- (n=4)WT littermates (n=2)
serum IgG Xab
IL-7 -/- WT
mea
n flu
ores
cenc
e in
tens
ity
0
100
200
300
400
500
IL-7 -/-
0
100
200
300
400
500day18 at rejection
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.
Survival hamster heart xenografts
days after transplantation
0 5 10 15 20 25 30 35 40 45 50 55 85 90 95 100
% s
urvi
val
0
20
40
60
80
100
IL-7 -/- (n=8)WT littermate (n=5)
IL-7 -/- given 90 x 106 IL-7 -/-splenocytes d-1 (n=1)
IL-7 -/- given 40 x 106 C57BL/6 splenocytes d0 (n=2)
IL-7 -/- given 40 x 106 C57BL/6 presensit splenocytes d+2 (n=2)
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)
Survival hamster heart xenografts
days after transplantation
0 5 10 15 20 25 30 35 40 45 50 55 85 90 95 100
% s
urvi
val
0
20
40
60
80
100
IL-7 -/- (n=8)
IL-7 -/- given 90 x 106 IL-7 -/-splenocytes d-1 (n=1)
IL-7 -/- given 40 x106 C57BL/6 splenocytes d0 (n=2)
IL-7 -/- given 40 x106 C57BL/6 splenocytes d-3 (n=2)
IL-7 -/- given 40 x106 C57BL/6 splenocytes d-6 (n=3)
IL-7 -/- given 40 x106 C57BL/6 splenocytes d-10 (n=2)
Results: NK xeno-cytotoxicity in IL-7 KO mice
NK cytotoxicity to ham ster blastce lls in s trains
0
10
20
30
40
50
60
E:T(40:1) E:T(20:1) E:T(10:1)
C57BL/6 WT mice Balb/c WT mice IL-7KO mice
Conclusion:
Functional NK xeno-cytotoxicity is normal in IL-7 KO mice
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 recipientson 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• 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.