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

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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

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30

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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.