Increased apoptosis of immunoreactive host cells and augmented donor leukocyte chimerism, not sustained inhibition of B7 molecule expression are associated with prolonged cardiac allograft survival in mice preconditioned with immature donor dendritic cells plus anti-CD40L mAb.
BACKGROUND
We previously reported the association among donor leukocyte chimerism, apoptosis of presumedly IL-2-deficient graft-infiltrating host cells, and the spontaneous donor-specific tolerance induced by liver but not heart allografts in mice. Survival of the rejection-prone heart allografts in the same strain combination is modestly prolonged by the pretransplant infusion of immature, costimulatory molecule-(CM) deficient donor dendritic cells (DC), an effect that is markedly potentiated by concomitant CM blockade with anti-CD40L (CD154) monoclonal antibody (mAb). We investigated whether the long survival of the heart allografts in the pretreated mice was associated with donor leukocyte chimerism and apoptosis of graft-infiltrating cells, if these end points were similar to those in the spontaneously tolerant liver transplant model, and whether the pretreatment effect was dependent on sustained inhibition of CM expression of the infused immature donor DC. In addition, apoptosis was assessed in the host spleen and lymph nodes, a critical determination not reported in previous studies of either spontaneous or "treatment-aided" organ tolerance models.


METHODS
Seven days before transplantation of hearts from B10 (H-2b) donors, 2x10(6) donor-derived immature DC were infused i.v. into C3H (H-2k) recipient mice with or without a concomitant i.p. injection of anti-CD40L mAb. Donor cells were detected posttransplantation by immunohistochemical staining for major histocompatibility complex class II (I-Ab) in the cells of recipient lymphoid tissue. CM expression was determined by two-color labeling. Host responses to donor alloantigen were quantified by mixed leukocyte reaction, and cytotoxic T lymphocyte (CTL) assays. Apoptotic death in graft-infiltrating cells and in areas of T-dependent lymphoid tissue was visualized by terminal deoxynucleotidyltransferase-catalyzed dUTP-digoxigenin nick-end labeling and quantitative spectrofluorometry. Interleukin-2 production and localization were estimated by immunohistochemistry.


RESULTS
Compared with control heart transplantation or heart transplantation after only DC administration, concomitant pretreatment with immature donor DC and anti-CD40L mAb caused sustained elevation of donor (I-Ab+) cells (microchimerism) in the spleen including T cell areas. More than 80% of the I-Ab+ cells in combined treatment animals also were CD86+, reflecting failure of the mAb to inhibit CD40/ CD80/CD86 up-regulation on immature DC in vitro after their interaction with host T cells. Donor-specific CTL activity in graft-infiltrating cells and spleen cell populations of these animals was present on day 8, but decreased strikingly to normal control levels by day 14. The decrease was associated with enhanced apoptosis of graft-infiltrating cells and of cells in the spleen where interleukin-2 production was inhibited. The highest levels of splenic microchimerism were found in mice with long surviving grafts (>100 days). In contrast, CTL activity was persistently elevated in control heart graft recipients with comparatively low levels of apoptotic activity and high levels of interleukin-2.


CONCLUSIONS
The donor-specific acceptance of rejection-prone heart allografts by recipients pretreated with immature donor DC and anti-CD40L mAb is not dependent on sustained inhibition of donor DC CM (CD86) expression. Instead, the pretreatment facilitates a tolerogenic cascade similar to that in spontaneously tolerant liver recipients that involves: (1) chimerism-driven immune activation, succeeded by deletion of host immune responder cells by apoptosis in the spleen and allograft that is linked to interleukin-2 deficiency in both locations and (2) persistence of comparatively large numbers of donor-derived leukocytes. These tolerogenic mechanisms are thought to be generic, explaining the tolerance induced by allografts spontaneously, or with the aid of various kinds of immunosuppression.
Lu
L
L
Thomas E. Starzl Transplantation Institute, and Department of Surgery, University of Pittsburgh, Pennsylvania 15213, USA.
Li
W
W
Zhong
C
C
Qian
S
S
Fung
J J
JJ
Thomson
A W
AW
Starzl
T E
TE
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015703:Antigens, CD / Q000096:biosynthesis*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000276:immunology; D017209:Apoptosis; D051940:B7-2 Antigen; D001854:Bone Marrow Cells / Q000276:immunology; D023201:CD40 Ligand; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D006085:Graft Survival / Q000502:physiology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D016027:Heart Transplantation / Q000276:immunology* / Q000473:pathology*; D016130:Immunophenotyping; D015847:Interleukin-4 / Q000494:pharmacology; D017708:Leukocyte Transfusion; D008221:Lymphoid Tissue / Q000166:cytology*; D008297:Male; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D013154:Spleen / Q000166:cytology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D018183:Transplantation Chimera / Q000276:immunology*; D019172:Transplantation Conditioning / Q000379:methods*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D051940:B7-2 Antigen; C497965:Cd86 protein, mouse; D008562:Membrane Glycoproteins; D016212:Transforming Growth Factor beta; D023201:CD40 Ligand; D015847:Interleukin-4; D016178:Granulocyte-Macrophage Colony-Stimulating Factor